Methods for the production of tcr gamma delta + t cells

ABSTRACT

The present invention relates to novel methods for the isolation and the selective ex vivo expansion of Vδ2 −  TCRγδ + T cells and to their clinical application.

FIELD OF THE INVENTION

The present invention relates to novel methods for the isolation and theselective ex vivo expansion of Vδ2⁻ TCRγδ⁺ T cells and to their clinicalapplication.

BACKGROUND TO THE INVENTION

TCRγδ⁺ T Cells

The immune system of jawed vertebrates includes various lymphocytepopulations capable of recognizing and eliminating tumor cells, whichconstitutes the basis of cancer immunotherapy. One population ischaracterized by the expression of a T-cell receptor (TCR) formed by thejunction of a gamma (γ) chain and a delta (δ) chain. TCRγδ⁺ T cells(here also designated as γδ T cells) account for 1-10% of humanperipheral blood lymphocytes (PBLs) but are substantially enriched inepithelial tissues of healthy individuals, where they reach up to 50% ofT cells.¹ TCRγδ⁺ T cells have potent Major Histocompatibility Complex(MHC)-unrestricted cytotoxicity against malignant and infected cells,while leaving unharmed healthy cells and tissues. Therefore, they areusually considered a first-line surveillance mechanism against infectionand tumors.¹

In humans, different subsets or subpopulations of TCRγδ⁺ T cells areidentified and classified based on the genes that encode their δ chain.Around 60-95% of TCRγδ⁺ T cells in the peripheral blood express the Vδ2chain in association with Vγ9 chain, while most of the remainder TCRγδ⁺T cells express the Vδ1 chain in association with various Vγ elements:Vγ2, Vγ3, Vγ4, Vγ5 or Vγ8. Other (rarer) human TCRγδ⁺ T cell populationsexpress Vδ3, Vδ5, Vδ6, Vδ7 and Vδ8 chains.²⁻⁴

Vδ2⁺ TCRγδ⁺ T Cells

Current adoptive immunotherapy approaches based on TCRγδ⁺ T cells arelimited to the Vδ2⁺ TCRγδ⁺ T cell subpopulation (here also designated asVδ2⁺ T cells).^(5, 6) Most Vδ2⁺ T cells specifically respond tononpeptide alkylphosphates such as isopentenyl pyrophosphate (IPP),which is produced at abnormal levels in tumor cells and in individualsexposed to bone-strengthening aminobisphosphonates, such as zoledronateand pamidronate. These compounds, when combined with interleukin-2,stimulate the proliferation and anti-tumour cytotoxic function of Vδ2⁺ Tcells in vitro, and generate purified cell populations for clinicalapplications.⁵⁻⁷ However, the clinical trials completed to date haveshown a low percentage of objective responses in cancer patients.⁸ Thus,current γδ T cellbased treatments, although feasible and safe, haveobvious limitations.⁶

Vδ1⁺ TCRγδ⁺ T cells

Human Vδ1⁺ TCRγδ⁺ T cells (here also designated as Vδ1⁺ T cells),constitute 1-40% of all TCRγδ⁺ PBLs but are the major γδ T cellpopulation at epithelial sites, such as the intestine and the skin.While it has never been evaluated in clinical trials, this cell subsetcan also display strong antitumor activity. Vδ1⁺ T cells infiltratingskin, colon, kidney and lung tumours were cytotoxic against bothautologous and allogeneic cancer cells.⁹⁻¹³ An interesting correlationwas also found between the increase in number of donor-derivedperipheral blood Vδ1⁺ T cells and improved 5-10-year disease-freesurvival following bone marrow transplantation for acute lymphoblasticleukaemia (ALL).¹⁴ Importantly, the infused Vδ1⁺ T cells persisted inthese patients for several years.^(14, 15) In another setting, low-gradenon-Hodgkin lymphoma patients with high Vδ1⁺ T cell counts experiencedstable disease at 1 year follow-up, an improved clinical course comparedto those with a lower number of Vδ1⁺ T cells.¹⁸ Circulating Vδ1⁺ T cellsare also typically increased in chronic lymphocytic leukemia (CLL)patients¹⁷ and have been associated with non-progression in low riskB-CLL patients.¹⁸ Finally, quantitative increases in circulating Vδ1⁺ Tcells were also observed in human immunodeficiency virus (HIV)¹⁹ andmalaria²⁰ infections, as well as in human cytomegalovirus (HCMV)infections following renal transplantation.^(4, 21) In differentcircumstances, however, subpopulations of Vδ1⁺ T cells can exhibitimmunosuppressive and regulatory properties, a function that can also beexploited for therapeutic purposes.²² A small number of methods tospecifically expand Vδ1⁺ T cells in vitro have been described, althoughnone of them could be adapted for clinical applications (reviewed inSiegers, G. et al., 2014, ref.²²). Meeh et al. first showed that Vδ1⁺ Tcells from healthy donors could expand ex vivo in response to ALLleukemic blasts.²³ Knight, A. and colleagues and Merims, S. andcolleagues isolated peripheral blood Vδ1⁺ T cells and treated them inthe presence of phytohemagglutinin (PHA) or anti-CD3 monoclonal antibody(mAb), IL-2 and irradiated allogeneic peripheral blood mononuclear cells(PBMCs), for 3 weeks.^(24, 25) In a more recent study, PHA was used incombination with interleukin-7 (IL-7).²⁶ Siegers, G. et al, reported atwo-step culture protocol, in which sorted TCRγδ⁺ T cells were firsttreated with concanavalin-A (ConA), IL-2 and IL-4 for 6-8 days, followedby stimulation with IL-2 and IL-4 for another 10 days.²⁷ After theculture period, Vδ1⁺ T cells were expanded in 59% of cultures and werethe dominant subset (average 70% of Vδ1⁺ T cells) in half of thecultures; however, a maximum 25 fold increase of Vδ1⁺ T cells could beachieved with this method.²⁷ This 2-step culture method was alsodescribed in International Patent Application Number PCT/CA99/01024(published as WO 00/26347). According to those inventors, for continuedcell proliferation following the removal of the mitogen, both IL-2 andIL-4 in the second culture medium were essential. A variation of thisprotocol was later developed, in which total PBMCs were first culturedin the presence of ConA, IL-2 and IL-4 for 6-13 days, followed by themagnetic depletion of contaminant TCRαβ⁺ T cells and stimulation of theremaining cells with IL-2, IL-4 and ConA for another 10 days. After 21days, Vδ1⁺ T cells expanded from 136 up to 24,384 fold, although a muchlower purity level could be achieved (less than 30% of cells in culturewere Vδ1⁺ T cells), while most contaminant cells (around 55% of cells)were Vδ2⁺ T cells.²⁸

Finally, our group has previously described a method to selectivelyexpand and differentiate cell populations enriched in Vδ1⁺ T cellsexpressing natural cytotoxicity receptors (NCRs) that could mediateimproved killing of leukemia cell lines and CLL patient neoplasticcells.²⁹ This patented method consisted in culturing TCRγδ⁺ T cells orprecursors thereof in a culture medium upon stimulation with commonγ-chain cytokines (such as IL-2 or IL-15) and TCR agonists (e.g., PHA oranti-CD3 mAb) for 2-3 weeks (International Patent Application NumberPCT/IB2012/052545 published as WO 2012/156958). One important limitationof this method is the small number of cells obtained, inappropriate forclinical applications.

Other Human TCRγδ⁺ T Cell Subsets

The majority of non-Vδ1⁺ and non-Vδ2⁺ TCRγδ⁺ T cells in humans expressthe Vδ3 TCR chain. Human TCRγδ⁺ Vδ3⁺ T cells account for ˜0.2% ofcirculating T cells,³⁰ but are enriched in the peripheral blood of renaland stem cell transplant recipients with CMV activation,^(4, 21) inpatients with HIV infection³¹ or with B-CLL,¹⁷ and in healthy livers.³²Activated TCRγδ⁺ Vδ3⁺ T cells were able to kill CD1d⁺ cells andepithelial tumors in vitro.⁴ However, available cell culture methodscannot produce large numbers of TCRγδ⁺ Vδ3⁺ T cells for clinicalapplications.

Other Methods for the Production of TCRγδ⁺ T Cell-Enriched Populations

Several methods have been used for the simultaneous expansion of severalsubsets of TCRγδ⁺ T cell populations in vitro. Total PBMCs were treatedwith plate-bound anti-TCRγδ mAb and IL-2 for 3 weeks, resulting in about90% of TCRγδ⁺ T cells, most of which were Vδ2⁺ T cells, and a smallpercentage of Vδ1⁺ T cells.³³ Lopez, et al, generatedapoptosis-resistant TCRγδ⁺ T cells by treating cells with high doseinterferon-γ (IFN-γ), IL-12, anti-CD2 mAb and soluble anti-CD3 mAb, inthe presence of IL-2. Vδ2⁺ T cells were the dominant subset after cellexpansion.³⁴ More recently, polyclonal TCRγδ⁺ T cells were expanded inthe presence of γ-irradiated artificial antigen-presenting cells (aAPC)of tumor origin genetically modified to co-express CD19, CD64, CD86,CD137L, and membrane bound IL-15.^(3,35) Cells were further cultured inthe presence of soluble IL-2 and IL-21.

Need for Improved Methods for Expanding Vδ2⁻ TCRγδ⁺ T Cells In Vitro

Recent studies have demonstrated that TCRγδ⁺ T cells with the Vδ1 chainand those with neither Vδ1 nor Vδ2 chains have properties which makesthem more attractive anticancer effectors in adoptive immunotherapy.³⁶Vδ2⁻ TCRγδ⁺ T cells (here also designated as Vδ2⁻ γδ T cells) exhibitedhigher anti-tumor cytotoxicity and increased survival capacities thanVδ2⁺ T cells, both in vitro and in vivo.^(26, 29, 37) Consequently, amedicinal product highly enriched in Vδ2⁻ γδ T cells is expected togenerate more potent anti-tumor effects and to provide improved benefitsto treated patients. While several methods have been described in thepast 5 years that can generate substantial numbers of tumor-targetingVδ2⁻ γδ T cells in vitro, key unresolved problems still excluded aclinical application of these cells: 1) the use of unsafe reagents andmaterials in the manufacturing process; 2) the high level of variationin the composition of the final cell products, especially of cellproducts obtained from different donors, or obtained from cancerpatients; and/or 3) the low anti-tumor activity of the finalproduct.^(22, 27, 28, 38) There is a need in the art for reliableclinical-grade cell culture methods that can generate a large number ofessentially pure Vδ2⁻ γδ T cells (i.e., comprising >90% of these cells)in vitro or ex vivo, consistently and with similar efficacy fromdifferent donors, especially from cancer patients.

Previous clinical data on infused Vδ2⁺ T cells suggested that aclinically relevant dose of TCRγδ⁺ T cells for treating a diseasecomprises at least about 1×10⁹ live cells.³⁹ Consequently, methodsaiming to generate Vδ2⁻ γδ T cells for clinical applications should beable to expand these cells in vitro by at least 1.000 fold. However,Vδ2⁻ γδ T cells do not respond to alkylphosphates and the very limitedknowledge about the antigens they recognize has limited their expansionin vitro. Plant lectins such as PHA and Con-A have been used to expandand enrich (to very high purity levels) these cells in vitro, at apre-clinical scale. Accordingly, and because of yet unknown reasons,these common mitogens are remarkably selective for Vδ2⁻ γδ T cells,promoting their proliferation in culture, while inhibiting growth (orinducing apoptosis) of contaminant Vδ2⁺ T cells.^(27, 29) Nevertheless,plant lectins can be toxic if inadvertently infused in humans andregulatory agencies do not recommend their use for clinicalapplications, due to safety concerns. Moreover, according to our data,plant lectins are not as efficient as monoclonal antibodies at inducingthe expansion of Vδ2⁻ γδ T cells in vitro, generating smaller numbers ofcells. Several groups have cultured TCRγδ⁺ T cells in the presence of ananti-CD3 monoclonal antibody, instead of PHA. However, the anti-CD3 mAbbinds to CD3 molecules also expressed on contaminant CD3⁺ TCRαβ⁺ T cellsand CD3+Vδ2⁺ T cells, causing a decrease in the purity levels of thefinal cell product. Consequently, Vδ2⁻ γδ T cells must be isolated bymagnetic-activated cell sorting (MACS) or by fluorescence-activated cellsorting (FACS) prior to stimulation with anti-CD3 mAb.^(25, 40) Tocomplicate things further, critical reagents such as the anti-TCRγδ mAb,anti-TCRVδ1 mAb and anti-TCRVδ3 mAb used to isolate total TCRγδ⁺ Tcells, TCRγδ+Vδ1⁺ T cells and TCRγδ+Vδ3⁺ T cells, respectively, are notcurrently manufactured or approved (by regulatory agencies) for clinicaluse. Therefore, sufficient numbers of purified Vδ2⁻ γδ T cells suitablefor direct application in humans have not been possible to generate.²²There is a need in the art for methods that do not rely on plant lectinsand other unsafe reagents for the production of purified Vδ2⁻ γδ T cellpopulations. In a previous study, Deniger et al. developed tumor-derivedartificial antigen presenting cells (aAPCs) to propagate high numbers ofTCRγδ⁺ T cells expressing a polyclonal repertoire of γ and δ TCRchains.³⁷ However, as pointed out by the authors,⁴¹ the method could notresolve critical obstacles associated with clinical application ofTCRγδ⁺ T cells. For example, most ingredients are not currently producedin GMP quality and further developments still depend on future interestof manufacturers, complex regulatory approvals, while assuming that thesame cell product can be obtained with different reagents, and fromcancer patients. Furthermore, key details regarding the exactcomposition (and variability) of the generated cell products weremissing in this study, thus hindering the potential application of thismethod.

Interleukin-2, interleukin-7 and interleukin-15 are very pleiotropicmolecules, with strong stimulatory effects on multiple immune cells,including TCRαβ⁺ T and Vδ2⁺ T cells. Consequently, these reagents arenot appropriate for expanding Vδ2⁻ γδ T cells in vitro, as contaminatingcells will also expand in culture, compromising cell purity.Furthermore, the typical combination of these pro-inflammatory cytokineswith γδTCR agonists often leads to activation-induced-cell-death (AICD)of stimulated cells and to smaller numbers of cells obtained. There is aneed in the art for methods that can rely on more selective reagents forexpanding Vδ2⁻ γδ T cells from highly impure starting samples.

The combination of IL-2 and IL-4 has been used with some success toexpand Vδ1⁺ T cells in vitro. However, we found that the presence ofIL-4 in the culture medium induces a strong downregulation of naturalkiller (NK) activating receptors (such as NKG2D and NCRs) on Vδ2⁻ γδ Tcells, weakening their anti-tumor responses (Table 3). The inhibitoryeffect of IL-4 on Vδ2⁻ γδ T cells occurred even when IL-2 was present.Along the same line, in an independent study, Mao, Y. and colleaguesrecently demonstrated that cultured Vδ1⁺ T cells treated with IL-4 andanti-TCRVδ1 mAb secreted significantly less IFN-γ and more IL-10relative to Vδ2⁺ T cells. Furthermore, IL-4-treated Vδ1⁺ T cellsexpressed lower levels of NKG2D, also indicating that IL-4 weakens theTCRγδ⁺ T cell-mediated anti-tumor immune response.⁴² These observations,together with recent findings on the tumor-promoting effects of Vδ1⁺ Tcells producing interleukin-17 (ref.^(43, 44)), have raised concernsabout their application, and stressed the need for a detailedcharacterization of effector Vδ1⁺ lymphocytes that might be consideredfor adoptive cell therapy. There is clearly a need in the art formethods able to expand Vδ2⁻ γδ T cells in vitro without theimmunosuppressive or inhibitory effects of IL-4.

Finally, several published methods aiming to expand Vδ2⁻ γδ T cells exvivo require the presence of natural or artificial feeder cells, usuallyin the form of virus-infected or transformed cells or cell lines,bacteria and parasites. These culture methods are more complex, moreprone to microbial contamination and less suitable for clinicalapplications.

SUMMARY OF THE INVENTION

The present invention provides novel methods for expanding anddifferentiating human Vδ2⁻ TCRγδ⁺ T cells in vitro, without the need forthe use of feeder cells or microbial or viral components. First, theinventors aimed at improving the expansion and purity levels of culturedVδ2⁻ TCRγδ⁺ T cells. A novel, more efficient and selective method toexpand these cells in culture was developed, in the presence of a T cellmitogen and IL-4, and in the absence of IL-2, IL-7 and IL-15. Finally,the obtained Vδ2⁻ TCRγδ⁺ T cells were differentiated towards a morecytotoxic phenotype through additional in vitro optimization steps.After the removal of IL-4 and the addition of a T cell mitogen andIL-15, IL-2, or IL-7 to the culture medium, the previously obtained Vδ2⁻TCRγδ⁺ T cells produced pro-inflammatory cytokines and expressed highlevels of activating Natural Killer receptors (NKR), which mediatedtumor cell killing in vitro. Importantly, upon infusion in mice, thedifferentiated TCRγδ⁺ T cells maintained their cytotoxic phenotype andinhibited tumor growth in vivo.

The cell culture method described herein is very robust, highlyreproducible and fully compatible with large-scale clinicalapplications. It generates sufficient numbers of differentiated Vδ2⁻TCRγδ⁺ T cells for use in adoptive immunotherapy of cancer, and in avariety of experimental, therapeutic and commercial applications.

Accordingly, in a first aspect, the present invention provides a methodfor expanding and differentiating Vδ2⁻ TCRγδ⁺ T cells in a samplecomprising:

(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and at least one growth factor having interleukin-4-likeactivity; in the absence of growth factors having interleukin-15-likeactivity; and

(2) culturing the cells obtained in step (1) in a second culture mediumcomprising a T cell mitogen and at least one growth factor havinginterleukin-15-like activity, in the absence of growth factors havinginterleukin-4-like activity.

Preferably, the present invention provides a method for expanding anddifferentiating Vδ2⁻ TCRγδ⁺ T cells in a sample comprising:

(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and interleukin-4; in the absence of interleukin-15,interleukin-2 and interleukin-7; and

(2) culturing the cells obtained in step (1) in a second culture mediumcomprising a T cell mitogen and interleukin-15, in the absence ofinterleukin-4.

The newly obtained method (detailed herein), is based on previouslyunidentified biological properties of Vδ2⁻ TCRγδ⁺ T cells and has notbeen described elsewhere.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel methods for the isolation and theselective in vitro/ex vivo expansion and differentiation of Vδ2⁻ TCRγδ⁺T cells, and to their clinical application. The inventors testedmultiple combinations of clinical-grade agonist antibodies and cytokinesfor their capacity to expand and differentiate (over 2-3 weeks)peripheral blood Vδ2⁻ TCRγδ⁺ T cells in culture. TCRγδ⁺ T cells wereisolated and expanded in culture in the absence of feeder cells andmolecules of microbial origin. The inventors have shown that Vδ2⁻ TCRγδ⁺T cells (here also designated as Vδ2⁻ γδ T cells), can be selectivelyexpanded in vitro by culturing these cells in a first culture mediumcomprising a T cell mitogen and interleukin-4, in the absence ofinterleukin-2, interleukin-7 and interleukin-15, and sub-culturing thesecells in a second culture medium containing a T cell mitogen andinterleukin-15, interleukin-2, or interleukin-7, in the absence ofinterleukin-4. Other key growth factors such as interferon-γ,interleukin-21 and interleukin-1β were also added to one or both culturemedia to further increase the expansion and purity levels of culturedVδ2⁻ γδ T cells.

The first culture medium supported the selective survival and expansionof Vδ2⁻ γδ T cells (up to 8.000-fold increase of Vδ1⁺ T cells in 14days; Table 3). Importantly, the absence of interleukin-2, interleukin-7and interleukin-15 during the first days of culture contributed to thestarvation and apoptosis of contaminant cells (including TCRαβ⁺ T andVδ2⁺ T cells), which critically depend on these cytokines for survival.

Finally, the presence of IL-2, IL-7, or IL-15 and the absence of IL-4 inthe second culture medium permitted the differentiation of thepreviously selected Vδ2⁻ γδ T cell population, which expanded in vitro atotal of several thousand fold and reached >90% of total cells after 21days of culture (Tables 3 and 7). This second culture step is necessaryto change the physiological properties of the cells towards a moreappropriate phenotype for use as an anti-tumor or anti-viral treatment.Expanded and differentiated Vδ2⁻ γδ T PBLs obtained after the secondculture step expressed high levels of activating Natural Killerreceptors (NKR), including NKp30 and NKp44, which synergized with theT-cell receptor to mediate tumor cell targeting in vitro, while nottargeting healthy cells. Infusion of the expanded and differentiatedVδ2⁻ γδ T PBLs cells in tumor-bearing immunodeficient mice inhibitedtumor growth and limited tumor dissemination to multiple organs,compared to non-treated animals. No evidence of treatment-associatedtoxicity in biochemical and histological analyses was found.Importantly, expanded and differentiated Vδ2⁻ γδ T PBLs were obtainedwith similar efficacy from blood samples of healthy donors and leukemiapatients. Finally, the present invention discloses methods for isolatingand culturing cells using materials and reagents fully compatible withindustrial and clinical applications. TCRγδ⁺ T cells were first sortedin a two-step protocol suitable to be used in a clinical-grade cellsorting machine (CliniMACS; Miltenyi Biotec, GmbH; Germany). Then, cellswere cultured with minimum manipulation in a serum-free cell culturemedium. Closed, large-scale, gas-permeable plastic cell culture bagswere used as recipients for cell culture, instead of open culture platesor flasks. All used reagents and materials (or equivalent reagents andmaterials) are currently available and manufactured in clinical-gradequality, or in Good Manufacturing Practice (GMP) quality, free ofanimal-derived components. Therefore, cells produced by this method canbe used in a variety of experimental, therapeutic and commercialapplications.

In a first aspect, the present invention provides a method for expandingand differentiating Vδ2⁻ TCRγδ⁺ T cells from a sample containing TCRγδ⁺T cells or precursors thereof, comprising:

(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and at least one growth factor having interleukin-4-likeactivity; in the absence of growth factors having interleukin-15-likeactivity; and

(2) culturing the cells obtained in step (1) in a second culture mediumcomprising a T cell mitogen and at least one growth factor havinginterleukin-15-like activity, in the absence of growth factors havinginterleukin-4-like activity.

The method of the first aspect of the invention results in expanded cellpopulations of Vδ2⁻ TCRγδ⁺ T cells. By “expanded” it is meant that thenumber of the desired or target cell type in the final preparation ishigher than the number in the initial or starting cell population.

The term “a T cell mitogen” means any agent that can stimulate T cellsthrough TCR signaling including, but not limited to, plant lectins suchas phytohemagglutinin (PHA) and concanavalin A (ConA) and lectins ofnon-plant origin, antibodies that activate T cells, and othernon-lectin/non-antibody mitogens. Preferred antibody clones includeanti-CD3 antibodies such as OKT-3 and UCHT-1 clones, anti-TCRγδantibodies such as B1 and IMMU510, or anti-TCRVδ1 antibodies such asδTCS1. Within the context of the present invention, antibodies areunderstood to include monoclonal antibodies (mAbs), polyclonalantibodies, antibody fragments (e.g., Fab, and F(ab′)2), single chainantibodies, single chain variable fragments (ScFv) and recombinantlyproduced binding partners. In one embodiment, the antibody is ananti-CD3 monoclonal antibody (mAb). Other mitogens include phorbol12-myristate-13-acetate (TPA) and its related compounds, such asmezerein, or bacterial compounds (e.g., Staphylococcal enterotoxin A(SEA) and Streptococcal protein A). The T cell mitogen may be soluble orimmobilized and more than one T cell mitogen may be used in the methodof the invention.

In the present invention, it was clearly demonstrated that two distinctgroups of cytokines, interleukin-4 on one hand, and interleukine-15,interleukin-2 and interleukin-7 on the other hand, must be used for veryspecific purposes in each culture step, and have opposite actions oncultured Vδ2⁻ γδ T cells. Based on the present study of the effects ofIL-4 and IL-15/IL-2/IL-7 on Vδ2⁻ γδ T cells, it should be obvious forany one skilled in the art that these two groups of cytokines arerepresentative members of two groups of growth factors, either having“interleukin-4-like activity” or “interleukin-15-like activity”, whichcan be used under the scope of the present invention.

The term “a growth factor having interleukin-4-like activity” means anycompound that has the same activity as IL-4 with respect to its abilityto promote similar physiological effects on Vδ2⁻ γδ T cells in cultureand includes, but is not limited to, IL-4 and IL-4 mimetics, or anyfunctional equivalent of IL-4. The physiological effects promoted byIL-4 on Vδ2⁻ γδ T cells (as described in the present invention), includethe decrease of NKG2D and NCR expression levels, the inhibition ofcytotoxic function and improved selective survival. Some of the referredactivities of IL-4 on Vδ2⁻ γδ T cells were also reported independentlyby another group. In that study, IL-4 significantly inhibited thesecretion of pro-inflammatory cytokines, including IFN-γ, TNF-α, fromactivated TCRγδ⁺ T cells.⁴⁵

The term “a growth factor having interleukin-15-like activity” means anycompound that has the same activity as IL-15 with respect to its abilityto promote similar physiological effects on Vδ2⁻ γδ T cells in cultureand includes, but is not limited to, IL-15 and IL-15 mimetics, or anyfunctional equivalent of IL-15, including IL-2 and IL-7. Thephysiological effects promoted by IL-15, IL-2 and IL-7 on cultured Vδ2⁻γδ T cells (as described in the present invention) were essentiallyequivalent, namely, the induction of cell differentiation towards a morecytotoxic phenotype, including the upregulation of NKG2D and NCR (NKp30and NKp44) expression levels, increased anti-tumor cytotoxic functionand increased production of pro-inflammatory cytokines, such as IFN-γ.

The terms “in the absence of interleukin-15, interleukin-2 andinterleukin-7” and “in the absence of interleukin-4” refer not only tothe complete absence of these cytokines in the culture medium, but alsoinclude the use of such cytokines at concentration levels so low thatthey cannot produce a measurable response or physiological effect intarget cells and thus can be considered absent for practical purposes.Furthermore, “a measurable physiological effect in target cells” refersto any measurable change in the cells' physiological state, within thescope of the present invention and according to standard definitions.For example, changes in the cell's physiological state can be detectedby changes in their activation state (recognized by the up-regulation ordownregulation of the expression levels of the early-activation cellmarker CD69); or detected by changes in their differentiation state(recognized by the up-regulation or downregulation of NKG2D or NCRs), afew hours or a few days after contact with such cytokines. A measurablephysiological effect may also be a change in the cell's proliferationrate, as measured by CFSE staining or by other techniques known in theart. It should be apparent for any one skilled in the art that cellscultured in the first culture medium must not receive a functionallyrelevant stimulus by IL-2, IL-7 and IL-15 or functionally similar growthfactors. Additionally, cells in the second culture medium must notreceive a functionally relevant stimulus by IL-4 or functionally similargrowth factors. Preferably, these cytokines must not be present in thecell culture medium at a final concentration higher than 2 ng/ml; morepreferably, not higher than 1 ng/ml, more preferably not higher than 0.1ng/ml, more preferably, they should be absent.

Preferably, the present invention provides a method for expanding anddifferentiating Vδ2⁻ γδ T cells in a sample comprising:

(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and interleukin-4; in the absence of interleukin-15,interleukin-2 and interleukin-7; and

(2) culturing the cells obtained in step (1) in a second culture mediumcomprising a T cell mitogen and interleukin-15, in the absence ofinterleukin-4.

In another embodiment, the present invention provides a method forexpanding and differentiating Vδ2⁻ γδ T cells in a sample comprising:

(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and interleukin-4; in the absence of interleukin-15,interleukin-2 and interleukin-7; and

(2) culturing the cells obtained in step (1) in a second culture mediumcomprising a T cell mitogen and interleukin-2, in the absence ofinterleukin-4.

Additionally, in another embodiment, the present invention provides amethod for expanding and differentiating Vδ2⁻ γδ T cells in a samplecomprising:

(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and interleukin-4; in the absence of interleukin-15,interleukin-2 and interleukin-7; and

(2) culturing the cells obtained in step (1) in a second culture mediumcomprising a T cell mitogen and interleukin-7, in the absence ofinterleukin-4.

The first or second culture medium, or both culture media, mayadditionally include other ingredients that can assist in the growth andexpansion of the Vδ2⁻ γδ T cells. Examples of other ingredients that maybe added, include, but are not limited to, plasma or serum, purifiedproteins such as albumin, a lipid source such as low density lipoprotein(LDL), vitamins, amino acids, steroids and any other supplementssupporting or promoting cell growth and/or survival.

The first or second culture medium, or both culture media, may alsocontain other growth factors, including cytokines that can furtherenhance the expansion of Vδ2⁻ γδ T cells. Examples of such cytokinesinclude, but are not limited to: (i) Interferon-γ and any growth factorhaving interferon-γ-like activity, (ii) interleukin-21 and any growthfactor having interleukin-21-like activity and (iii) IL-1β and anygrowth factor having interleukin-1β-like activity. Examples of othergrowth factors that can be added include co-stimulatory molecules suchas a human anti-SLAM antibody, any soluble ligand of CD27, or anysoluble ligand of CD7. Any combination of these growth factors can beincluded in the first or second culture medium, or in both media.

The term “a growth factor having interferon-γ-like activity” means anycompound that has the same activity as IFN-γ with respect to its abilityto promote survival or proliferation of Vδ2⁻ γδ T cells in culture andincludes, but is not limited to, IFN-γ and IFN-γ mimetics, or anyfunctional equivalent of IFN-γ.

The term “a growth factor having interleukin-21-like activity” means anycompound that has the same activity as IL-21 with respect to its abilityto promote survival or proliferation of Vδ2⁻ γδ T cells in culture andincludes, but is not limited to, IL-21 and IL-21 mimetics, or anyfunctional equivalent of IL-21.

The term “a growth factor having interleukin-1β-like activity” means anycompound that has the same activity as IL-1β with respect to its abilityto promote survival or proliferation of Vδ2⁻ γδ T cells in culture andincludes, but is not limited to, IL-1β and IL-1β mimetics, or anyfunctional equivalent of IL-1β.

In particular, the inventor has found that the addition of a secondgrowth factor having interferon-γ-like activity to the first or secondculture medium, or to both culture media, resulted in enhanced expansionof Vδ2⁻ γδ T cells as compared to the expansion obtained using onegrowth factor.

Accordingly, in one embodiment, the method of the first aspect of theinvention comprises:

-   -   (1) culturing cells in the sample in a first culture medium        comprising a T cell mitogen, interleukin-4 and interferon-γ, in        the absence of interleukin-2, interleukin-7 and interleukin-15;        and    -   (2) culturing the cells obtained in step (1) in a second culture        medium comprising a T cell mitogen, interleukin-15 and        interferon-γ, in the absence of interleukin-4, to expand and        differentiate Vδ2⁻ γδ T cells.

Preferably, the growth factor having IFN-γ-like activity is present inan amount from about 1 to about 1000 ng/ml. More preferably, this growthfactor is present in an amount from about 2 to about 500 ng/ml. Morepreferably, this growth factor is present in an amount from about 20 toabout 200 ng/ml. Most preferably, the second culture medium comprisesabout 70 ng/mL of a growth factor having IFN-γ-like activity, such asIFN-γ.

The inventor has also found that the addition of a second growth factorhaving IFN-γ-like-activity and a third growth factor havingIL-21-like-activity to the first or second culture medium, or to bothculture media, resulted in enhanced expansion of Vδ2⁻ γδ T cells ascompared to the expansion obtained using one or two growth factors.

Accordingly, in one embodiment, the method of the first aspect of theinvention comprises:

-   -   (1) culturing cells in the sample in a first culture medium        comprising a T cell mitogen, interleukin-4, interferon-γ and        interleukin-21, in the absence of interleukin-2, interleukin-7        and interleukin-15; and    -   (2) culturing the cells obtained in step (1) in a second culture        medium comprising a T cell mitogen, interleukin-15 and        interferon-γ, in the absence of interleukin-4, to expand and        differentiate Vδ2⁻ γδ T cells.

Preferably, the growth factor having IL-21-like-activity is present inan amount from about 1 to about 500 ng/ml. More preferably, this growthfactor is present in an amount from about 2 to about 200 ng/ml. Morepreferably, this growth factor is present in an amount from about 5 toabout 100 ng/ml. Most preferably, the second culture medium comprisesabout 15 ng/mL of a growth factor having IL-21-like-activity, such asIL-21.

The inventor has also found that the addition of a second growth factorhaving IFN-γ-like-activity, and a third growth factor havingIL-21-like-activity, and a fourth growth factor having IL-1β-likeactivity, to the first or second culture medium, or to both culturemedia, resulted in enhanced expansion of the Vδ2⁻ γδ T cells as comparedto the expansion obtained using one, two or three growth factors.

Accordingly, in one embodiment, the method of the first aspect of theinvention comprises:

-   -   (1) culturing cells in the sample in a first culture medium        comprising a T cell mitogen, interleukin-4, interferon-γ,        interleukin-21 and interleukin-1β, in the absence of        interleukin-2, interleukin-7 and interleukin-15; and    -   (2) culturing the cells obtained in step (1) in a second culture        medium comprising a T cell mitogen, interleukin-15, interferon-γ        and interleukin-21, in the absence of IL-4, to expand Vδ2⁻ γδ T        cells.

Preferably, the growth factor having IL-1β-like-activity is present inan amount from about 1 to about 500 ng/ml. More preferably, this growthfactor is present in an amount from about 2 to about 200 ng/ml. Morepreferably, this growth factor is present in an amount from about 5 toabout 100 ng/ml. Most preferably, the second culture medium comprisesabout 15 ng/mL of a growth factor having IL-1β-like-activity, such asIL-1β.

The inventor has also found that the addition of a co-stimulatorymolecule to the first or second culture medium, or to both culturemedia, resulted in enhanced expansion of the Vδ2⁻ γδ T cells as comparedto the expansion obtained without using such molecule.

Accordingly, in one embodiment, the method of the first aspect of theinvention comprises:

-   -   (1) culturing cells in the sample in a first culture medium        comprising a T cell mitogen, interleukin-4, and any molecular        ligand of CD27, or any molecular ligand of SLAM or any molecular        ligand of CD7 receptors, in the absence of interleukin-2,        interleukin-7 and interleukin-15; and    -   (2) culturing the cells obtained in step (1) in a second culture        medium comprising a T cell mitogen and interleukin-15, in the        absence of IL-4, to expand Vδ2⁻ γδ T cells.

The term “a molecular ligand” means any molecule or compound that bindsto a specific target receptor. In particular, the inventor has foundthat the addition of a soluble ligand of CD27, or a soluble ligand ofCD7 or a soluble ligand of SLAM resulted in enhanced expansion of Vδ2⁻γδ T cells. These soluble ligands constitute functional agonists of eachone of these molecular receptors, and any similar agonists binding tothese receptors can induce the same effect on Vδ2⁻ γδ T cells, forexample, agonistic antibodies such as human anti-SLAM antibodies, humananti-CD27 antibodies and human anti-CD7 antibodies.

More than one subculture step can be performed during the total cultureperiod. For example, each of the previously described subculture stepscan be further divided into two subculture steps (1a) and (1b) and (2a)and (2b), and different combinations of ingredients can be usedaccording to the originally described method.

Accordingly, in one embodiment, the method of the first aspect of theinvention comprises:

-   -   (1) culturing cells in the sample in a first culture medium        comprising a T cell mitogen, interleukin-4, interferon-γ,        interleukin-1β and interleukin-21, in the absence of        interleukin-2, interleukin-7 and interleukin-15; and    -   (2a) culturing the cells obtained in step (1) in a second        culture medium comprising a T cell mitogen, interleukin-15 and        interleukin-21, in the absence of interleukin-4; and    -   (2b) culturing the cells obtained in step (2a) in a third        culture medium comprising a T cell mitogen, interleukin-15 and        interferon-γ, in the absence of interleukin-4, to expand Vδ2⁻ γδ        T cells.

In another embodiment, the method of the first aspect of the inventioncomprises:

-   -   (1a) culturing cells in the sample in a first culture medium        comprising a T cell mitogen, interleukin-4, interferon-γ, and        interleukin-21, in the absence of interleukin-2, interleukin-7        and interleukin-15; and    -   (1 b) culturing the cells obtained in step (1a) in a second        culture medium comprising a T cell mitogen, interleukin-4,        interferon-γ, and interleukin-1β, in the absence of        interleukin-2, interleukin-7 and interleukin-15; and    -   (2) culturing the cells obtained in step (1 b) in a third        culture medium comprising a T cell mitogen and interleukin-15,        in the absence of interleukin-4; to expand Vδ2⁻ γδ T cells.

The TCRγδ⁺ T cells obtained by the method of the invention can be usedin a variety of experimental, therapeutic and commercial applications.This includes, but is not limited to, subsequent genetic modification orgenetic editing of such cells, for example with the objective ofimproving their therapeutic potential. For example, with the objectiveof redirecting the specificity of the TCRγδ⁺ T cells through theexpression of a chimeric antigen receptor (CAR) or TCR on these cells.CAR expression can be induced through electroporation of TCRγδ⁺ cellsfor the insertion of genetic material, or by infecting these cells withviral vectors, such as lentiviruses or retroviruses containing thedesired genetic material. Such genetic editing may improve the potencyof the TCRγδ⁺ T cells by improving homing, cytokine production, recyclekilling, and/or improved engraftment.

The present invention provides novel methods for selectively expandingVδ2⁻ TCRγδ⁺ T cells in culture. The methods of the first aspect of theinvention are carried out on a sample, which is also referred to hereinas a “starting sample”. The methods can use either unfractionatedsamples or samples which have been enriched for TCRγδ⁺ T cells.

The sample can be any sample that contains TCRγδ⁺ T cells or precursorsthereof including, but not limited to, blood, bone marrow, lymphoidtissue, epithelia, thymus, liver, spleen, cancerous tissues, lymph nodetissue, infected tissue, fetal tissue and fractions or enriched portionsthereof. The sample is preferably blood including peripheral blood orumbilical cord blood or fractions thereof, including buffy coat cells,leukapheresis products, peripheral blood mononuclear cells (PBMCs) andlow density mononuclear cells (LDMCs). In some embodiments the sample ishuman blood or a fraction thereof. The cells may be obtained from asample of blood using techniques known in the art such as densitygradient centrifugation. For example, whole blood may be layered onto anequal volume of Ficoll-Hypaque™ followed by centrifugation at 400×g for15-30 minutes at room temperature. The interface material will containlow density mononuclear cells which can be collected and washed inculture medium and centrifuged at 200×g for 10 minutes at roomtemperature.

Isolated or unpurified TCRγδ⁺ T cells can be cultured or maintained inany suitable mammalian cell culture medium such as AIM-V™, RPMI 1640,OPTMIZER CTS™ (Gibco, Life Technologies), EXVIVO-10, EXVIVO-15 orEXVIVO-20 (Lonza), in the presence of serum or plasma. Cells can betransferred, for example, to VueLife® clinical-grade gas-permeable cellculture static bags (Saint Gobain) or to Miltenyi Biotec'sclinical-grade cell culture bags. Cell bags containing cells and culturemedium can be placed in an incubator at 37° C. and 5% CO₂, in the dark.

Prior to culturing the sample or fraction thereof (such as PBMCs) in thefirst culture medium, the sample or fraction thereof may be enriched forcertain cell types and/or depleted for other cell types. In particular,the sample or fraction thereof may be enriched for T cells, or enrichedfor TCRγδ⁺ T cells, or depleted of TCRαβ⁺ T cells or depleted ofnon-TCRγδ⁺ T cells. In a preferred embodiment, the sample is firstdepleted of TCRαβ⁺ T cells and then enriched for CD3+ cells.

The sample may be enriched or depleted of certain cell types usingtechniques known in the art. In one embodiment the cells of a particularphenotype may be depleted by culturing the sample or fraction thereofwith an antibody cocktail containing antibodies that bind to specificmolecules on the cells to be depleted. Preferably, the antibodies in thecocktail are coupled to magnetic microbeads that can be used tomagnetically deplete or enrich target cells when these cells are forcedto pass through a magnetic column.

Once the cells in the sample have been fractionated and enriched, ifdesired, the cells are cultured in a first culture medium comprising a Tcell mitogen and at least one growth factor having interleukin-4-likeactivity, such as interleukin-4, in the absence of growth factors havinginterleukin-15-like activity, such as interleukin-15, interleukin-2 andinterleukin-7.

Preferably, the T cell mitogen in the first culture medium is present inan amount from about 10 to about 5000 ng/ml. More preferably, the T cellmitogen is present in an amount from about 20 to about 2000 ng/ml. Morepreferably, the T cell mitogen is present in an amount from about 50 toabout 1000 ng/ml. Most preferably, the medium comprises 70 ng/mL of a Tcell mitogen.

Preferably, the growth factor having interleukin-4-like activity ispresent in an amount from about 1 to about 1000 ng/ml. More preferably,this growth factor is present in an amount from about 5 to about 500ng/ml. More preferably, this growth factor is present in an amount fromabout 20 to about 200 ng/ml. Most preferably, the medium comprises 100ng/mL of a growth factor having interleukin-4-like activity, such asIL-4.

The cells are preferably cultured in the first culture medium for aperiod of time ranging from about 2 days to about 21 days. Morepreferably, from about 3 days to about 14 days. More preferably, fromabout 4 days to 8 days.

Following culture in the first culture medium, cells are sub-cultured ina second culture medium comprising a T cell mitogen and at least onegrowth factor having interleukin-15-like activity, such as IL-15, IL-2or IL-7, in the absence of growth factors having interleukin-4-likeactivity, such as IL-4. If the cells are sub-cultured, for example, inthe presence of both IL-15 and IL-4, then proliferation continues butcell viability decreases and key NK receptors located at the cellsurface (such as NKG2D, NKp30 and NKp44) are internalized to theinterior of the cell. Consequently, these receptors can no longer bindto their ligands expressed on tumor cells, which reduces the anti-tumorcytotoxic activity of these cells.

The subculture step consists in culturing the cells obtained in step 1in a new culture medium. This can be achieved through the addition offresh culture medium to the first culture medium, preferably after theremoval of a fraction of the first culture medium. This can be done bycentrifuging and/or decanting the cells, removing a fraction of thefirst culture medium and resuspending the cells in the second culturemedium. Preferably, the subculture step involves the removal of at least¾ of the first culture medium. The subculture step should be carried outbecause it is important for the expansion and differentiation of Vδ2⁻ γδT cells that the growth factor having interleukin-4-like activity isremoved during the subculture step.

Preferably, in the second culture medium, the T cell mitogen is presentin an amount from about 0.1 to about 50 μg/ml. More preferably, the Tcell mitogen is present in an amount from about 0.3 to about 10 μg/ml.More preferably, the T cell mitogen is present in an amount from about0.5 to about 5 μg/ml. Most preferably, the medium comprises 1 μg/mL of aT cell mitogen.

Preferably, the growth factor having interleukin-15-like activity, suchas IL-15, IL-2 or IL-7, is present in an amount from about 1 to about1000 ng/ml. More preferably, this growth factor is present in an amountfrom about 2 to about 500 ng/ml. More preferably, this growth factor ispresent in an amount from about 20 to about 200 ng/ml. Most preferably,the second culture medium comprises about 70 ng/mL of a growth factorhaving interleukin-15-like activity, such as IL-15, IL-2 or IL-7.

The cells are preferably cultured in the second culture medium for aperiod of time ranging from about 2 days to about 30 days. Morepreferably, from about 5 days to about 21 days. More preferably, fromabout 10 days to 15 days.

Preferably, in the first aspect of the invention, both the first andsecond culture media are supplemented with serum or plasma. The amountof plasma in the first and second culture media is preferably from about0.5% to about 25% by volume, for example from about 2% to about 20% byvolume or from about 2.5% to about 10% by volume, for example is about5% by volume. The serum or plasma can be obtained from any sourceincluding, but not limited to, human peripheral blood, umbilical cordblood, or blood derived from another mammalian species. The plasma maybe from a single donor or may be pooled from several donors. Ifautologous TCRγδ⁺ T cells are to be used clinically, i.e. reinfused intothe same patient from whom the original sample was obtained, then it ispreferable to use autologous plasma as well (i.e. from the same patient)to avoid the introduction of hazardous products (e.g. viruses) into thatpatient. The plasma should be human-derived to avoid the administrationof animal products to the patient.

The TCRγδ⁺ T cells obtained according to the method of the first aspectof the invention can be separated from other cells that may be presentin the final culture using techniques known in the art includingfluorescence activated cell sorting, immunomagnetic separation, affinitycolumn chromatography, density gradient centrifugation and cellularpanning.

TCRγδ⁺ T cells obtained by the method of the first aspect of theinvention are also of use. Accordingly, the inventor describes a cellpreparation of TCRγδ⁺ T cells.

In a second aspect, the present invention provides a cell preparationenriched in TCRγδ⁺ T cells prepared according to the method of the firstaspect of the invention.

In a third aspect, the present invention provides a cell preparationenriched in TCRγδ⁺ T cells wherein greater than 80% of the total cellsare TCRγδ⁺ T cells.

Preferably, in the second and third aspects of the invention the TCRγδ⁺T cells comprise greater than 80%, more preferably greater than 90% andmost preferably greater than 95%, of the total cells in the enrichedpopulation.

The TCRγδ⁺ T cells obtained by the method of the first and second aspectof the invention may be used in any and all applications. TCRγδ⁺ T cellsare thought to be a first line of defense against infectious pathogens.In addition, TCRγδ⁺ T cells possess intrinsic cytolytic activity againsttransformed cells of various origins including B-cell lymphomas,sarcomas and carcinomas. As a result, the TCRγδ⁺ T cells obtained andcultured ex vivo according to the methods of the invention can betransfused into a patient for the treatment or prevention of infections,cancer or diseases resulting from immunosuppression.

Accordingly, in a fourth aspect the present invention provides a methodof modulating an immune response comprising administering an effectiveamount of TCRγδ⁺ T cells prepared according to a method of the first orsecond aspect of the invention or obtained from a cell preparationaccording to the second or third aspect of the invention to an animal inneed thereof.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desiredresults.

The term “animal” as used herein includes all members of the animalkingdom. Preferably, the animal is a mammal, more preferably a human.

In a fifth aspect, the present invention provides a method of treatingan infection comprising administering an effective amount of TCRγδ⁺ Tcells prepared according to the method of the first or second aspect ofthe invention or obtained from a cell preparation according to thesecond or third aspect of the invention to an animal in need thereof.

Examples of infections that may be treated include, but are not limitedto, bacterial infections such as those caused by Mycobacteria (e.g.tuberculosis), viral infections such as those caused by herpes simplexvirus (HSV), human immunodeficiency virus (HIV) or the hepatitisviruses, and parasitic infections such as those caused by Plasmodium(e.g. malaria).

In a sixth aspect, the present invention provides a method for treatingcancer comprising administering an effective amount of TCRγδ⁺ T cellsprepared according to the method of the first or second aspect of theinvention or obtained from a cell preparation according to the second orthird aspect of the invention to an animal in need thereof.

Examples of cancer that may be treated include, but are not limited to,leukemias including chronic lymphocytic leukemia, chronic myelogenousleukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, andT cell and B cell leukemias, lymphomas (Hodgkin's and non-Hodgkins),lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas,adenomas, sarcomas, carcinomas of solid tissues, hypoxic tumors,squamous cell carcinomas, genitourinary cancers such as cervical andbladder cancer, hematopoietic cancers, head and neck cancers, andnervous system cancers.

In one embodiment, the cancer to be treated is chronic lymphocyticleukemia. In such an embodiment, PBMCs can be obtained from a patientwith chronic lymphocytic leukemia (CLL). After culturing and expandingfor TCRγδ⁺ T cells, the expanded cells will not significantly containcancerous CLL cells making them well suited for re-infusion back to thepatient.

These aspects of the invention also extend to the TCRγδ⁺ T cellsobtained by a method of the first or second aspect of the invention orobtained from a cell preparation according to the second or third aspectof the invention for use in a method of modulating an immune response,treating an infection or treating cancer as described herein above. Theinvention further includes the use of the TCRγδ⁺ T cells obtainedaccording to methods of the first or second aspect of the invention inthe manufacture of a medicament or pharmaceutical composition tomodulate an immune response, to treat an infection or to treat cancer asdescribed hereinabove.

The TCRγδ⁺ T cells obtained according to the present invention can alsobe used in experimental models, for example, to further study andelucidate the function of the cells. Additionally, these cells may beused for studies directed towards the identification of theantigens/epitopes recognized by TCRγδ⁺ T cells and for the design anddevelopment of vaccines.

Accordingly, in a seventh aspect, the present invention provides amethod for vaccinating an animal comprising administering an effectiveamount of TCRγδ⁺ T cells obtained by a method of the first aspect of theinvention or obtained from a cell preparation according to the second orthird aspect of the invention to an animal in need thereof. Such vaccinecan be given to immunocompromised patients or individuals with elevatedrisk of developing an infectious disease or cancer.

This aspect of the invention also extends to the use of TCRγδ⁺ T cellsprepared according to the first aspect of the invention or obtained froma cell preparation according to the second or third aspect of theinvention for the manufacture of a vaccine, and to TCRγδ⁺ T cellsprepared according to the method of the first aspect of the invention orobtained from a cell preparation according to the second or third aspectof the invention for use in a method of vaccinating an animal.

The obtained TCRγδ⁺ T cells, according to the invention may beimmediately used in the above therapeutic, experimental or commercialapplications or the cells may be cryopreserved for use at a later date.

Preferred features of the second and subsequent aspects of the inventionare as described for the first aspect of the invention mutatis mutandis.

Other features and advantages of the present invention will be apparentfrom this detailed description. It should be understood, however, thatthe detailed description and the specific examples while indicatingpreferred embodiments of the invention are given by way of illustrationonly, since various changes and modifications will become apparent tothose skilled in the art from this detailed description.

The present invention is completely different from other inventions thatwere previously described in this field. Patent Application n^(o)PCT/CA1999/001024 (WO/2000/026347; Filing Date of 4 Nov. 1999),describes a method for the production of TCRγδ⁺ T cells. The methodinvolves two steps, wherein TCRγδ⁺ T cells in the starting sample arecultured in a first culture medium comprising a T cell mitogen and atleast two cytokines, preferably interleukin-2 and interleukin-4. Cellsobtained in the first step are then cultured in a second culture mediumcomprising at least two cytokines, which are preferably interleukin-2and interleukin-4. Importantly, cytokines used in each step can be thesame or different. In contrast, the present invention discloses a 2-stepmethod for selectively expanding and differentiating Vδ2⁻ γδ T cells inculture, wherein the first and second culture steps are necessarilydifferent from each other. The first step comprises culturing TCRγδ⁺ Tcells in the starting sample in a culture medium comprising a T cellmitogen and a growth factor having IL-4-like activity, preferablyinterleukin-4, in the absence of interleukin-2, interleukin-7 andinterleukin-15. In the present invention, it was clearly demonstratedthat IL-4 and IL-2 (or IL-7 or IL-15) execute a very specific functionin each culture step, and have opposite activities. The first culturemedium contains a growth factor having IL-4-like activity, which cannotbe mixed with (IL-2/IL-7/IL-15-like) growth factors, otherwise cellviability and proliferation will decrease and the expected cell productwill not be produced. The first cell culture step is used to expandTCRγδ⁺ T cells many fold in an exceptional short period of time,generating a cell product highly enriched in Vδ2⁻ γδ T cells. In fact,the absence of IL-2/IL-7-IL15 like growth factors in the culture mediumallows the elimination of many impurities (other cell types) that wouldnot be eliminated without this innovation. The expanded Vδ2⁻ γδ T cellsare in a less differentiated state since they have not contactedinterleukin-2/-15/-7-like growth factors. In order to differentiatecells towards a more cytotoxic and pro-inflammatory phenotype, Vδ2⁻ γδ Tcells obtained in the first step must then be cultured in a secondculture medium comprising a T cell mitogen and a growth factor havinginterleukin-15-like activity, in the absence of a growth factor havinginterleukin-4-like activity. Again, it is critical that these differentgrowth factors are not mixed, since the presence of IL-4 in the secondculture medium decreased cell viability and cytotoxic function. Thesecond culture step further expands Vδ2⁻ γδ T cells in culture and makesthem better effector cells, generating a distinctive cell product thatcan be used for many therapeutic purposes.

Patent n^(o) US2003/0157060 A1 (Application n^(o) U.S. Ser. No.10/239,854); and Filing Date of 3 Mar. 2000), describes another methodfor expanding TCRγδ⁺ T cells in culture. The method also involves twosteps, wherein TCRγδ⁺ T cells in the starting sample are cultured in afirst culture medium comprising (1) a T cell mitogen, (2) a growthfactor having interleukin-2-like activity and (3) a growth factor havinginterleukin-7-like activity. Cells obtained in the first step are thencultured in a second culture medium comprising (1) a growth factorhaving interleukin-2-like activity and a (2) growth factor havinginterleukin-7-like activity to expand TCRγδ⁺ T cells.

In contrast, the present invention is different since the two culturesteps are necessarily different, as they serve distinct purposes. Theinvention clearly excludes the use of growth factors havinginterleukin-2-like activity or interleukin-7-like activity in the firstculture step, due to reasons already explained (see above). Although thepresent invention describes the presence of other growth factors in thefirst and/or second culture medium, including cytokines such asInterferon-γ, IL-1β and IL-21, these cytokines cannot be considered tohave interleukin-2-like or interleukin-7-like activity since thephysiological effects that they exert on Vδ2⁻ γδ T cells are verydifferent. For example, cells cultured in the presence of Interferon-γ,IL-1β and IL-21 (in the absence of IL-2, IL-7 and IL-15) could notacquire their full cytotoxic function and differentiated state, thusInterferon-γ, IL-1β and IL-21 could not mimic the high pro-inflammatoryeffects induced by IL-2, IL-7 and IL-15 on cultured Vδ2⁻ γδ T cells(Table 2). Additionally, some of these cytokines, such as Interferon-γand IL-1β, belong to a structurally different family of cytokines.

The invention will now be described with reference to the followingExamples and Figures, in which:

FIGURES

FIG. 1 shows percentages of TCRγδ⁺ T PBLs in a peripheral blood samplecollected from a healthy donor, before and after magnetic activated cellsorting (MACS). 50-150 ml of fresh peripheral blood was obtained from ahealthy volunteer and diluted in a 1:1 ratio (volume-to-volume) with PBS(Invitrogen Gibco) and centrifuged in Ficoll-Paque (Histopaque-1077;Sigma-Aldrich) in a volume ratio of 1:3 (1 part ficoll to 3 parts ofdiluted blood) for 35 minutes at 1.500 rpm and 25° C. The interphasecontaining mononuclear cells (PBMCs) was collected and washed (in PBS).Total TCRγδ⁺ T cells were labeled with an anti-TCRγδ mAb conjugated withmagnetic microbeads and TCRγδ⁺ T cells were isolated (to above 75%purity) with a magnetic column (Miltenyi Biotec, German). Cells werelabeled with the following fluorescent monoclonal antibodies:anti-CD3PerCP-Cy5.5 (Biolegend; clone SK7); anti-TCR-Vδ1-APC (MiltenyiBiotec, clone REA173); anti Mouse IgG1κ-APC Isotype Ctrl (MiltenyiBiotec; clone IS5-21F5). Cells were analyzed on a Fortessa II flowcytometer (BD Biosciences). Shows representative results of δindependent experiments.

FIG. 2 shows percentages of TCRγδ⁺ T PBLs in a peripheral blood samplecollected from the previously selected healthy donor, before and afterthe 2-steps MACS sorting procedure. 50-150 ml of fresh peripheral bloodwas obtained from healthy volunteers and diluted in a 1:1 ratio(volume-to-volume) with PBS (Invitrogen Gibco) and centrifuged inFicoll-Paque (Histopaque-1077; Sigma-Aldrich) in a volume ratio of 1:3(1 part ficoll to 3 parts diluted blood) for 35 minutes at 1.500 rpm and25° C. The interphase containing mononuclear cells (PBMCs) was collectedand washed (in PBS). Unwanted TCRαβ⁺ T lymphocytes were then labelled byincubation in the presence of a murine anti-human TCRαβ monoclonalantibody (mAb) conjugated to Biotin (Miltenyi Biotec Ref#701-48, cloneBW242/412). Cells were then labelled again with a murine anti-Biotin mAbcoupled to magnetic microbeads (Miltenyi Biotec Ref#173-01). Finally,the cell suspension was loaded onto a magnetic column (Miltenyi Biotec,Germany) and TCRαβ⁺ T lymphocytes were magnetically depleted (anddiscarded). CD3+ cells (of which most of them were TCRγδ⁺ T cells)present in the remaining cell population were labelled with a murineanti-human CD3 mAb (Miltenyi Biotec Ref#273-01, clone OKT-3, targetingan epitope located on the CD3epsilon chain), conjugated tosuperparamagnetic iron dextran particles. Cells were loaded onto amagnetic MACS separation column and CD3+ cells were positively selected(purified). Cells were stained for TCRγδ, CD3, TCRVδ1 and TCRVδ2markers, and analysed on a Fortessa II flow cytometer (BD Biosciences).Shows representative results of δ independent experiments.

FIG. 3 shows a summary of tested culture conditions. A-C. TCRγδ⁺ PBLsfrom a healthy donor were isolated by MACS as previously described andcultured in 96-well plates at 1 million cells/ml in complete medium(Optmizer CTS, GIBCO) supplemented with 5% human serum, 1 mML-glutamine, at 37° C. and 5% CO₂. Cells were equally distributed bymultiple wells and cultured in the presence of three fixed combinationsof anti-CD3 mAb and IL-4, further supplemented with variousconcentrations of IL-2, IL-7, IL-15 or IFN-γ. Fresh medium supplementedwith the same growth factors was added every 5-6 days. At the end of theculture period, cells were counted and cell phenotype was analyzed byflow cytometry. Each growth factor was used separately in serialdilutions from 500 ng/ml to 0.1 ng/ml. Graphs show the best culturecondition (i.e., highest fold expansion of Vδ1+ cells) obtained, foreach cytokine (IL-2, IL-7, IL-15 and IFN-γ). The control samplecontained IL-4 and anti-CD3 mAb only. D. Total CD3⁺ PBLs were isolatedby MACS with an anti-human CD3 mAb (coupled with paramagnetic beads)from the peripheral blood of the same donor and cultured and analyzed aspreviously described. Shows average±SD of 3 technical replicates.Statistical analysis was performed using Graphpad-Prism software.Differences between subpopulations were assessed using the Student ttest and are indicated when significant as *P<0.05; **P<0.01; and***P<0.001 in the figures.

FIG. 4 shows the percentages of Vδ1⁺ T PBLs before and after in vitroculture for 15 days. TCRγδ⁺ PBLs from a healthy donor were isolated byMACS as previously described and cultured in the presence of 200 ng/mlIFN-γ, 1 ug/ml α-CD3 and 100 ng/ml IL-4. A fraction of fresh mediumcontaining the same combination of growth factors was added every 5-δdays. Shows the FACS-plot analysis at day 15. Representative results of3 independent experiments.

FIG. 5 shows a panel of tested cell culture media. TCRγδ⁺ T PBLs from ahealthy donor were isolated by MACS as previously described and culturedin different commercially available serum-free, clinical grade cellculture media. Cells were cultured in a first culture medium in thepresence of 70 ng/ml IFN-γ, 1 μg/ml α-CD3 and 100 ng/ml IL-4, followedby culture in a second culture medium in the presence of 100 ng/ml IL-15and 2 μg/ml anti-CD3 mAb, (in the absence of IL-4). Shows final numberof cells and percentages of cells after FACS analysis at day 15. Showsaverage±SD of 3 technical replicates.

FIG. 6 shows that Vδ1⁺ T cells expand in vitro to become the dominantcell subset in culture. 70 ml of concentrated peripheral blood(corresponding to 450 ml of peripheral blood) was obtained from 8 BuffyCoat units collected from 8 healthy donors. Blood was centrifugedundiluted in Ficoll-Paque (Histopaque-1077; Sigma-Aldrich) in a volumeratio of 1:3 (1 part ficoll to 3 parts of blood) for 35 minutes at 1.600rpm and 25° C. The interphase containing mononuclear cells (PBMCs) wascollected and washed (in saline buffer). TCRγδ⁺ PBLs were isolated bythe previously described 2-step MACS and resuspended in serum-freeculture medium (OPTMIZER, GIBCO) supplemented with 5% autologous plasmaand 1 mM L-glutamine. Cells were seeded at a concentration of 0.5×10^(δ)cells/ml and expanded in closed gas-permeable, 1 L cell culture plasticbags in the incubator at 37° C. and 5% CO₂. Growth factors were added tothe cell culture media according to the previously described 2-stepprotocol: 70 ng/ml anti-CD3 mAb, 100 ng/ml IL-4, 70 ng/ml IFN-γ, 7 ng/mlIL-21 and 15 ng/ml IL-1β were added to the first culture medium and 70ng/ml IL-15, 100 ng/ml IFN-γ, 15 ng/ml IL-21 and 1 μg/ml anti-CD3 mAbwere added to the second culture medium. A fraction of old medium wasremoved and fresh medium was added every 5-δ days, supplemented withgrowth factors. Accessory “feeder” cells were not required in thissystem. Left panel shows percentages of CD3+Vδ1+ cells among total livecells as analyzed by flow cytometry. Right panel shows fold increase inthe absolute number of CD3+Vδ1⁺ T cells relative to the initial cellnumber. Live cells were counted using Trypan Blue-positive exclusion ina haemocytometer. Shows an average of 3 measurements of number of cells

FIG. 7 shows the expression of several cell surface markers in in vitroexpanded Vδ1⁺ T cells. Expression of the activating receptors NKp30,NKp44 and NKG2D in CD3+Vδ1⁺ T cells after 16 days of culture (as inTable 8). Isotype mAb control stainings are also shown (note that thecontrol for NKp30 and NKp44 expression is the same).

FIG. 8 shows that in vitro expanded TCRγδ⁺ T cells are cytotoxic againstCLL cells but not against autologous PBMCs. After 21 days of expansionand differentiation (as described in FIG. 6), the resulting TCRγδ⁺ Tcells were co-incubated with target cells (pre-labelled with DDAO-SE)for 3 h at 37° C., and target cell death was assessed by Annexin Vstaining. (A) Flow cytometry analysis of susceptible MEC-1 CLL cells(upper panel) and non-susceptible autologous PBMCs (lower panel).Representative plots of 3 technical replicates. (B) Impact of differenttarget:effector cell ratios and of blocking antibodies against NKG2D orNKp30 on MEC-1 leukemia cell killing. Error bars represent SD (n=3,*P<0.05). (C) Expanded (and differentiated) TCRγδ⁺ T cells (designatedherein as “DOT-cells”) of donor A were co-incubated with three B-CLLprimary cell samples (collected from the peripheral blood of CLL/SLLpatients and enriched for CD19 by MACS) or with autologous healthyPBMCs. Shows Mean+SD of 3 technical replicates.

Detailed methods: The MEC-1 CLL cell line⁴⁶ was obtained from the GermanResource Center for Biologic Material (DSMZ). MEC-1 tumor cells werecultured in T25 flasks in complete 10% RPMI 1640 with 10% Fetal BovineSerum, 2 mM L-Glutamine and maintained at 10⁵ up to 10^(δ) cells/mL bydilution and splitting in a 1:3 ratio every 3-4 days. For cytotoxicityassays, in vitro expanded TCRγδ⁺ T cells were plated in 96-wellround-bottom plates. Tumor cell lines or leukemia primary samples werestained with CellTrace Far Red DDAO-SE (1 μM; Molecular Probes,Invitrogen) and incubated at the indicated target:effector ratio withTCRγδ⁺ T cells in RPMI 1640 medium for 3 hours at 37° C. and 5% CO₂, inthe presence of 70 ng/ml IL-15. All cells were then stained with AnnexinVFITC (BD Biosciences) and analyzed by flow cytometry.

FIG. 9 shows that activated TCRγδ⁺ T cells produce high levels of IFN-γ.TCRγδ⁺ T cells were produced from two healthy donors in cell culturebags for 21 days, following the 2-step culture protocol, as previouslydescribed. Cells were washed, plated in 96-well plate and re-stimulatedwith fresh medium supplemented with 100 ng/ml IL-15 and 2 μg/ml anti-CD3mAb. After 48 h, cell culture supernatants were analyzed by flowcytometry by Cytometric bead array (BD Biosciences). OpTmizer refers tocells kept in media not supplemented with activating compounds. ShowsMean+SD of 3 technical replicates. Error bars represent SD (n=3,**P<0.01).

FIG. 10 shows that TCRγδ⁺ T cells from CLL/SLL patients expand robustlyin vitro and are highly cytotoxic against CLL/SLL cells and CMV-infectedcells. MACS-sorted TCRγδ⁺ PBLs from three CLL/SLL patients were culturedfor 21 days with cytokines and mAb as previously described. Left panel:Cells were analysed by flow cytometry for TCRVδ1/CD3 co-expression andfold increase of CD3⁺ Vδ1⁺ T cells was calculated. Live cells wereidentified by flow cytometry with a viability dye (Zombie Violet;Biolegend) and counted with Trypan Blue in a haemocytometer. Rightpanel: TCRγδ⁺ T cells from a CLL/SLL patient were co-cultured for 3 h at1:10 target-effector ratio in 96-well plate with CLL/SLL-derived MEC-1cells or with human foreskin fibroblasts (FFB), either healthy orpreviously infected with YFP⁺ Cytomegalovirus strain AD169 (m.o.i 0.005;error bars represent SD (n=3 for each group). Percentage of dead tumorcells before incubation with DOT-cells was around 20% in each group. SLLis for Small Lymphocytic Lymphoma.

FIG. 11 shows that in vitro expanded TCRγδ⁺ T cells are cytotoxicagainst cancer cells of diverse tissue origins. Peripheral blood TCRγδ⁺T lymphocytes from one healthy donor were MACS-sorted and stimulated exvivo in the presence of cytokines and anti-CD3 mAb for 21 days,according to the described two-step culture protocol. Cells wereco-incubated for 3 h in 96-well plates with a panel of tumor cell lines:MOLT-4 (Acute lymphoblastic leukemia; ALL); HL-60 (Acute myeloidleukemia; AML); K562 (Chronic myeloid leukemia; CML); MEC-1 (Chroniclymphocytic leukemia; CLL); HELA (Cervical carcinoma); Daudi (Burkitt'slymphoma); THP-1 (Acute monocytic leukemia; AMoL); MDA-231 (Breastcarcinoma), PA-1 (Ovarian carcinoma); PC3 (Prostate carcinoma) andHCT11δ(Colon carcinoma). Tumor cell death was evaluated by Annexin-Vstaining (n=3 technical replicates).

FIG. 12 shows that infused TCRγδ⁺ T cells home and survive in xenografttumor models. A. 2×10⁷ TCRγδ⁺ T cells were transferred intotumor-bearing (MEC-1 CLL\SLL cell-line) NSG immunodeficient hosts. 30days after transfer of TCRγδ⁺ T cells, animals were sacrificed andTCRγδ⁺ T cell-progeny was evaluated by FACS in the indicated tissues.Note that TCRγδ⁺ T cells were present in all tissues analyzed and alsothat CD3⁺ Vδ1⁺ T cells were highly enriched, suggesting preferentialfitness and/or activation in presence of CLL tumor. Dot-Plots are from arepresentative animal of δ animals analyzed. B. Infused TCRγδ⁺ T cellsexpress NCRs in vivo. TCRγδ⁺ T cells recovered from the liver at day 30after transfer were analyzed by FACS for NCR expression. Note highexpression of NKp30 and NKG2D. Top dot plots are isotype controls. Arepresentative animal of 3 animals analyzed is shown. C. Infused TCRγδ⁺T cells are able to home into BRG immunodeficient hosts. 10⁷ TCRγδ⁺ Tcells from a different donor were transferred into CLL tumor-bearing BRGhosts and quantified by FACS 72 hours after transfer. TCRγδ⁺ T cellscould be recovered from both the lung and liver and TCRVδ1⁺-expressingcells were found at proportions similar to the initial transfer. Oneanimal out of two is shown.

FIG. 13 shows that TCRγδ⁺ T cells can limit tumour growth in vivo. 10⁷Luciferase-expressing MEC-1 CLL/SLL tumour cells were transferredsubcutaneously into immunodeficient BRG hosts. 10 and 15 days aftertransfer, 10⁷ TCRγδ⁺ T cells or control PBS were transferred i.v. intoCLL tumour bearing hosts, as verified by luminescence analysis. CLL/SLLtumour growth was periodically measured using a calliper. Tumour size isshown, note the effect of TCRγδ⁺ T cells visible in CLL tumour size (n=8mice per group).

FIG. 14 shows that TCRγδ⁺ T cells limit CLL tumour spread. A. 10⁷Luciferase-expressing MEC-1 CLL/SLL tumour cells were transferredsubcutaneously into immunodeficient NSG hosts. 10 and 15 days aftertransfer, TCRγδ⁺ T cells (2×10⁷) or control PBS were transferred i.v.into CLL/SLL tumour bearing hosts, as verified by luminescence analysis.CLL/SLL tumour growth was periodically measured using a Caliper. Tumoursize is shown, note the partial and transient tendency early upontreatment but TCRγδ⁺ T cells were not able to limit the faster bulkCLL/SLL tumour growth in these hosts. B-D. The CLL/SLL tumour line isable to spread to other organs in these experiments. Plots shown in Bdepict FACS analysis of organs recovered at the end of the experimentfrom NSG hosts receiving TCRγδ⁺ T cells transfer or control PBS. Weobserved a generalized reduction in CLL/SLL tumour cells recovered indifferent organs but this effect was more pronounced in the liver, amajor organ for tumour spreading in this model. C. histological (H&E)analysis of a representative animal from each group, showing CLL/SLLtumour metastasis in the indicated anatomical sites of PBS treatedanimals and absence of tumour infiltrates in treated animals. (*p<0.05;**p<0.01).D. Summary of histological analysis is shown, depictinganimals from each group where CLL/SLL tumour infiltrates were foundagainst animals free of tumour infiltrates. Scoring was performedblindly by a certified pathologist on H&E stained histological samplesfrom the indicated tissues from animals in experiment depicted in FIG.15. Results are shown for all animals analysed and show a clearreduction in overall tumour spread in the group of animals treated within vitro expanded TCRγδ⁺ T cells.

FIG. 15 shows that TCRγδ⁺ T cells can infiltrate primary tumour and areactivated in vivo. A. Depicted is IHC analysis of CD3-stained sectionsof primary CLL/SLL tumour from NSG animals transferred with MEC-1 tumourcells and treated with TCRγδ⁺ T cells. B. We analysed CD69 (n=3, arepresentative dot-plot is shown), Nkp30 and NKG2D expression (n=2) invivo in different organs and found recently activated TCRγδ⁺ T cells,with major expression of these activation markers in TCRγδ⁺ T cellsrecovered from the CLL/SLL tumour.

FIG. 16 shows biochemical analysis of blood samples. (A) Blood wascollected at the time of necropsy from animals from experiment shown inFIG. 14 and biochemical analysis performed for Alanin-Aminotransferase(ALT/GPT), Aspartate Aminotransferase (AST/GOT), Blood Urea Nitrogen(BUN), Creatinin, Creatin phosphokinase (CPK) and Lactate Dehiidrogenase(LDH). (B) The same as in A, for animals shown in FIG. 11. We found noevidence for toxicity associated with DOT cell treatment.

DETAILED METHODS OF THE IN VIVO STUDIES

Balb/c Rag^(−/−)γc^(−/− 47) animals were obtained from Taconic (USA);NOD-SCIDγc^(−/− 48) mice were obtained from the Jackson Laboratories(USA). BRG or NSG mice were injected subcutaneously with MEC-1 cells andtreated after δ and 11 days with two intravenous transfers of 10⁷ or2×10⁷ DOT cells, and then analyzed (tumor size, histology, flowcytometry of tumor or organ infiltrates, and blood biochemistry) asdetailed. All animal procedures were performed in accordance to nationalguidelines from the Direção Geral de Veterinária and approved by therelevant Ethics Committee. For phenotyping after in vivo DOT-celltransfer, animals were euthanized using Eutasil in order for bloodcollection via cardiac puncture; and quickly perfused with PBS+Heparin.Organs were homogenized and washed in 70 μM cell strainers. Femurs wereflushed and then filtered. Cells were then stained with the followingantibodies from ebioscience, Biolegend, Myltenyi Biotec or BecktonDickinson: anti-mouse CD45 (30-F11), and anti-Human: CD45 (H130). Otherantibodies used are common with the in vitro studies. Antibodies werecoupled to FITC, PE, PerCP, PerCP-Cy5, PE-Cy7, APC, APC-Cy7, PacificBlue, Brilliant Violet 421 and Brilliant Violet 510 fluorochromes.Statistical analysis was performed using Graphpad-Prism software. Samplemeans were compared using the unpaired Student's t-test. In casevariances of the two samples were found different using F-test, the datawas log transformed and if variances were then found not to bedifferent, the unpaired t-test was applied to the log-transformed data.For Survival data, log-rank (Mantel-Cox) test was used.

In vivo experimental design: We used a previously described model ofxenografted human CLL upon subcutaneous adoptive transfer ofCLL/SLL-derived MEC-1 cells into Balb/cRag^(−/−)γc^(−/−) (BRG) animals,which we further adapted using NOD-SCIDγc^(−/−) (NSG) animals as hosts.In order to ensure that animals receiving treatment or PBS control weretumor-bearing animals, we transduced MEC-1 CLL cells withfirefly-luciferase in order to detect and measure tumor engraftment atearly time-points before ascribing treatment cells. After 7 or 4 days(in different studies) we injected luciferin i.p. to determine tumourload as a function of luminescence, before ascribing treatment (or PBScontrol) to the animals. Animals were distributed randomly in cages andassigned to each treatment (PBS or DOT-Cells) according to luminescencemeasured at day 7, in such a way that animal with highest luminescencereceived treatment, second highest received PBS, third highest receivedtreatment, etc. This resulted in a non-randomized distribution intogroups but randomized distribution in the different cages. 2 additionalanimals received DOT-Cells in the indicated experiments for initialhoming analysis. We performed two 10⁷ or 2×10⁷ DOT-Cells transfers(within 5 days), using cells from one different donor per experiment.Tumor was measured using a Caliper and taking three perpendicularmeasurements. The formula used was ½×L×W×H.⁴⁹ Animals were sacrificedwhen tumor measurements reached 1000 mm³.

Luminescence Analysis: After transduction of MEC-1 Cell line withGFP-firefly luciferase, growing cells were screened and sorted accordingto GFP expression using a FACS-Aria (Becton Dickinson, USA), up to >95%GFP positive cells. These cells were then kept in culture untiltransferred subcutaneous into host animals (in 50 μl PBS). At theindicated time points after transfer, animals were anesthetized(Ketamin/Medetomidine) and Luciferin was injected (i.p.). 4 min laterluciferase activity was detected and acquired using IVIS Lumina(Calliper LifeSciences) at the IMM bioimaging facilities. Anesthesia wasthen reverted and animals returned to previous housing.

Lymphocyte counts: Cell counts were performed with a hemocytometer orusing Accuri Flow cytometer (Becton Dickinson, USA). Counts per organwere estimated when parts of the organ were sampled for histologicalanalysis by weighting organs before and after the samples were split.Numbers presented are then corrected for the whole organ. In Bone Marrowdata, absolute numbers were calculated and are displayed for one femur.Histopathology and Immunohistochemistry: Mice were sacrificed withanesthetic overdose, necropsies were performed and selected organs(lung, heart, intestine, spleen, liver, kidney, reproductive tract,brain, cerebellum, spinal cord, and femur) were harvested, fixed in 10%neutral-buffered formalin, embedded in paraffin and 3 μm sections werestained with hematoxylin and eosin (H&E). Bones were further decalcifiedin Calci-Clear™ (Fisher Scientific) prior to embedding. Tissue sectionswere examined by a pathologist, blinded to experimental groups, in aLeica DM2500 microscope coupled to a Leica MC170 HD microscope camera.Immunohistochemical staining for CD3 (Dako, cat. no. A0452) wasperformed by the Histology and Comparative Pathology Laboratory at theIMM, using standard protocols, with a Dako Autostainer Link 48. Antigenheat-retrieval was performed in DAKO PT link with low pH solution (pH6), and incubation with ENVISION kit (Peroxidase/DAB detection system,DAKO, Santa Barbara, Calif.) was followed by Harri's hematoxylincounterstaining (Bio Otica, Milan, IT). Negative control included theabsence of primary antibodies; and CD3 staining was not observed in thenegative controls. Images were acquired in a Leica DM2500 microscope,coupled with a Leica MC170 HD microscope camera.

Mouse Blood Biochemistry: Mice were deeply anaesthetised and blood wascollected from the heart to heparin-coated tubes, sent for analysis ofbiochemical parameters shown at an independent laboratory. Biochemicalparameters were measured in serum, in a RX monaco clinical chemistryanalyser (RANDOX).

FIG. 17 shows TCRγδ+PBL enrichment after two-step MACS-sorting.

Peripheral blood (obtained from buffy coats) was collected from 4healthy volunteers, and total TCRγδ⁺ T cells were isolated by MACS:first, TCRαβ depletion was performed and then CD3⁺ cells were positivelyselected. Cells were stained for TCRγδ, CD3 and TCRVδ1 and analyzed byflow cytometry. Dot plots show fractions of TCRγδ⁺ PBLs before and aftereach MACS-sorting step (left panels); and initial and final percentagesof Vδ1⁺ T cells (right panels).

FIG. 18 shows the characterization of the activation and maturationphenotype of the obtained Vδ/1⁺ T cells.

(A) Flow cytometry comparison of the cell surface phenotype of Vδ1⁺ Tcells at day 21 of culture (using the previously described 2-stepculture protocol); (full lines) with freshly-isolated Vδ1⁺ T cells(dotted lines), as analyzed using the LEGENDScreen kit (Biolegend).Shown are histogram overlays for several markers related to lymphocyteactivation and differentiation, and markers implicated in adhesion andmigration. Cells from one healthy donor are shown. (B) Heatmaprepresenting percentages of positive cells for each surface markeracross cultured Vδ1⁺ T cells (at day 21 of culture) produced from 4different healthy donors (Cultur. 1-4), compared to freshly-isolatedVδ1⁺ T cells (from donors 1 and 2). The color code is presented on theright. For phenotyping after cell production: cells were stained withanti-CD3-APC (clone UCHT1), anti-TCRVδ1-FITC and a panel of receptorsusing the LegendScreen kit (Biolegend).

FIG. 19 shows TCR/NCR-dependent cytotoxicity of TCRγδ⁺ T cells againstleukemic (but not healthy) cells.

Expanded and differentiated TCRγδ⁺ T cells produced from two healthydonors (using the previously described 2-step culture protocol), weretested in different experiments against MEC-1 (CLL) target cells atincreasing effector/target ratios (left plot, gray bars) and also inpresence of blocking antibodies for (a, anti-) the indicated molecules,either individually (Expt 1) or in combinations (Expt 2). The highestEffector/Target ratio (10:1) was used in blocking experiments and graybar at this ratio (with IgG isotype antibody) serves as control. Shownare the percentages of dead (Annexin-V⁺) MEC-1 target cells. * and #indicate significant differences relative to IgG isotype control orα-TCRVδ1, respectively (Mean+SD; *, ^(#)p<0.05; **, ^(##)p<0.01;Student's t-test).

For cytotoxicity assays, MEC-1 tumor cells were cultured in T25 flasksin complete 10% RPMI 1640 with 10% Fetal Bovine Serum, 2 mM L-Glutamineand maintained at 10⁵ up to 10^(δ) cells/mL by dilution and splitting ina 1:3 ratio every 3-4 days. In vitro expanded TCRγδ⁺ T cells were platedin 96-well round-bottom plates. Tumor cells were stained with CellTraceFar Red DDAO-SE (1 μM; Molecular Probes, Invitrogen) and incubated atthe indicated target:effector ratio with TCRγδ⁺ T cells in RPMI 1640medium for 3 hours at 37° C. and 5% CO₂, in the presence of 70 ng/mlIL-15. For receptor blocking, γδ PBLs were pre-incubated for 1 hour withblocking antibodies: human anti-TCRγδ (clone B1); human anti-NKG2D(clone 1D11); human anti-CD2 (clone RPA-2.10); human anti-CD3 (cloneOKT-3); human anti-NKp30 (clone P30-15); human anti-NKp44 (clone P44-8),mouse IgG1,k (clone MOPC-21), mouse IgG2b (clone MPC-11), mouse IgG3k(clone MG3-35), all from Biolegend. Human anti-CD22δ(clone DX11) wasfrom BD Biosciences. Human anti-Vδ1 TCR (clones TCS-1 or TS8.2) werefrom Fisher Scientific, and human anti-TCRγδ (clone IMMU510) was from BDBiosciences. The blocking antibodies were maintained in the culturemedium during the killing assays. Finally, all cells were then stainedwith Annexin V-FITC (BD Biosciences) and analyzed by flow cytometry.

FIG. 20 shows the expression of activating cell surface NK receptors inin vitro expanded Vδ1⁺ and Vδ1⁻ Vδ2⁻ T cells. Expanded anddifferentiated TCRγδ⁺ T cells were produced from healthy donors, usingthe previously described 2-step culture protocol. Shows expression ofthe activating receptors NKp30 and NKp44 in CD3⁺ Vδ1⁺ cells and in CD3⁺Vδ1⁻ Vδ2⁻ T cells after 21 days of culture. First, CD3⁺ Vδ2⁺ cells wereidentified (with anti-CD3 and anti-Vδ2 mAbs conjugated to fluorochromes)and were excluded from the analysis. The remaining CD3+ cells wereanalyzed for the expression of Vδ1 and NKp30, NKp44 and Isotype Control.Shows representative results of 4 independent experiments with 4different donors.

FIG. 21 shows cytotoxicity of the expanded Vδ1⁻ Vδ2⁻ T cell subset ofTCRγδ⁺ T cells against leukemic cells.

Expanded and differentiated TCRγδ⁺ T cells produced from one healthydonor (using the previously described 2-step culture protocol) werestained for CD3, Vδ1 and Vδ2 T cell markers with monoclonal antibodiesconjugated to fluorochromes, and the CD3⁺ Vδ1⁻ Vδ2⁻ T cell populationwas isolated by flow cytometry. Isolated cells were then tested in akilling assay in vitro against acute myeloid leukemia (AML) target celllines (KG-1, THP-1, HL-60 and NB-4) at an effector/target ratio of 10:1.The killing assay was performed as previously described.

Tables:

Table 1 describes the molecules used to stimulate the proliferation ofVδ1⁺ T cells during the optimization stage. Reagents were used at aconcentration range from 0.1 ng/ml to 80 μg/ml. The column on the rightshows reagent distributor or manufacturer.

Table 2 shows a summary of tested culture conditions. TCRγδ⁺ PBLs wereisolated by MACS from a healthy donor and cultured at 1 million cells/mlin 96 well plates, at 37° C. and 5% CO₂. Cells were expanded in completemedium (Optmizer CTS, GIBCO) supplemented with 5% autologous plasma, 1mM L-glutamine and with the described growth factors. At the end of theculture period, cells were counted and cell phenotype was analyzed byflow cytometry. Shows selected results of 4 consecutive experiments. Thebest culture conditions in each experiment are ranked by fold increase.Fold expansion rate of Vδ1⁺ T cells was calculated as: (absolute numberof Vδ1⁺ T cells at the end of the culture)/(absolute number of Vδ1⁺ Tcells at day 0 of culture). Shows representative results of 2independent experiments.

Table 3 shows a summary of tested culture conditions. TCRγδ⁺ PBLs wereisolated by MACS from a healthy donor and cultured for 14 days in thepresence of the described growth factors. At day 14 of culture, cellswere split: one fraction of cells was cultured as before, while theother fraction of cells was cultured in the absence of IL-4 and in thepresence of the indicated growth factors. At day 21, cells were countedand cell phenotype was analyzed by FACS. Shows representative resultsfrom 2 independent experiments. A cytotoxicity assay was also performedat day 21 using the generated TCRγδ⁺ cells against MOLT-4 leukemiatargets (method is described in FIG. 8). Apoptotic (dying) target cellswere detected by positive staining with Annexin-V reagent in a FortessaII flow cytometry machine (BD Biosciences). Basal tumor cell death(i.e., the percentage of apoptotic tumor cells in tumor samples thatwere not co-cultured with TCRγδ⁺ cells) was 10±3%. Shows average of twotechnical replicates.

Table 4 shows a summary of tested culture conditions. TCRγδ⁺ PBLs wereisolated by MACS from a healthy donor and cultured for 15 days in atwo-step and three-step culture protocols, in the presence of thedescribed growth factors. Cells were counted and cell phenotype wasanalyzed by FACS at the end of the culture period. Shows representativeresults of 2 independent experiments.

Table 5 shows a summary of tested culture conditions. TCRγδ⁺ PBLs wereisolated by MACS from a healthy donor in a two-step protocol andcultured for 21 days in the presence of the described growth factors.Cells were counted and cell phenotype was analyzed by FACS at the end ofthe culture period. Shows representative results of 2 independentexperiments

Table δ shows the purity and phenotype of TCRγδ⁺ PBLs isolated via atwo-step MACS protocol. PBMCs were obtained by density gradientcentrifugation in ficoll from Buffy Coat products collected from 8healthy donors. TCRγδ⁺ T cells were further isolated by a two-step MACSprotocol as described in FIG. 2. Cell phenotype was characterized byflow cytometry analysis of cell surface antigens. Data correspond topercentages of total live cells.

Table 7 shows the purity and phenotype of in vitro expanded TCRγδ⁺ Tcells. MACS-sorted TCRγδ⁺ T cells from healthy donors (same as in Table6) were cultured for 21 days in cell culture bags, according to thepreviously described two-step culture protocol. Cell populations werecharacterized by flow cytometry. Indicates percentages of TCRγδ⁺ T cellsand contaminant cells, relative to total live cells present in thecultures.

Table 8 shows the expression of NCRs and NKG2D in freshly-isolatedversus cultured Vδ1⁺ T cells. Expression of the activating receptorsNKp30, NKp44 and NKG2D in CD3⁺ Vδ1⁺ T cells after 16 or 21 days ofcytokine and anti-CD3 mAb treatment. Data in this table arerepresentative of data obtained from 10 independent donors, noting thatNKp30 and NKp44 expression varied between around 10% and 70% amongdifferent donors, while NKG2D was expressed by more than 80% of Vδ1⁺PBLs of all tested donors.

Table 9 shows the purity and phenotype of pre- and post-MACS-sortedTCRγδ⁺ PBLs from CLL/SLL patients. B-cell chronic lymphocytic leukemia(CLL) cells were obtained from the peripheral blood of patients at firstpresentation, after informed consent and institutional review boardapproval. TCRγδ⁺ T cells were MACS-sorted from the peripheral blood of 3CLL patients (CLL-1-3) and cell population phenotype was characterizedby flow cytometry analysis of cell surface antigens. Shows percentagesof TCRγδ⁺ T cells and contaminant cells, obtained immediately before andafter the 2-step magnetic isolation procedure. Each cell subset wasgated on total live cells.

Table 10 shows that contaminant autologous B-CLL cells become a residualpopulation in culture. TCRγδ⁺ T cells were MACS-sorted from theperipheral blood of 3 CLL/SLL patients (CLL-1-3; as in Table 9) andcultured in vitro for 16 days as previously described. Cell populationphenotype was characterized by flow cytometry analysis of cell surfaceantigens. Shows percentages of TCRγδ⁺ T cells and contaminant cells.Each cell subset is gated on total live cells, except NKp30 and NKG2Dexpression that were gated on Vδ1⁺ T cells.

Table 11 shows in more detail the tested culture conditions presented inTable 2 of a previous application. TCRγδ⁺ PBLs were isolated by MACSfrom a healthy donor and cultured at 1 million cells/ml in 96 wellplates, at 37° C. and 5% CO₂. Cells were expanded in complete medium(Optmizer CTS, GIBCO) supplemented with 5% autologous plasma, 1 mML-glutamine and with the described growth factors. At the end of theculture period, cells were counted and cell phenotype was analyzed byflow cytometry. Shows selected results of 4 consecutive experiments (thesame experiments described in Table 2 of a previous application, butfurther discloses results of parallel control culture conditions, markedwith an asterisk, for a more complete understanding of the results). Italso shows the percentage of NKp30⁺ Vδ1⁺ T cells obtained with eachcondition. Culture conditions in each experiment were ranked by foldincrease. Fold expansion rate of Vδ1⁺ T cells was calculated as:(absolute number of Vδ1⁺ T cells at the end of the culture)/(absolutenumber of Vδ1⁺ T cells at day 0 of culture). Shows representativeresults of 2 independent experiments.

Table 12 shows a summary of tested culture conditions.

TCRγδ⁺ PBLs were isolated and expanded from a healthy donor, asdescribed previously, in the presence of the indicated growth factors.Shows selected results of one experiment with multiple cultureconditions. To better understand the effects of IL15/IL-2/IL-7 and IFN-γon cultured TCRγδ⁺ cells, TCRγδ⁺ cells were cultured in culture mediumand three different concentrations of IL-4 and anti-CD3 mAb, in thepresence or absence of IL15/IL-2/IL-7 and IFN-γ. Shows the detrimentaleffect of IL-15, IL-2 and IL-7 on TCRγδ⁺ T cell expansion, when thesecells were cultured in the presence of IL-4 and IFN-γ. Showsrepresentative results of 2 independent experiments.

Table 13 shows the total absolute number of TCRγδ⁺ cells obtained beforeand after the large-scale 2-step cell culture protocol. MACS-sortedperipheral blood TCRγδ⁺ cells obtained from healthy donors (representedin FIG. 6) were counted with a haemocytometer before and after cellculture expansion/differentiation. Each Buffy Coat was concentrated from450 ml of peripheral blood and contained around 450-550 million PBMCs.On average, 17 million TCRγδ⁺ T cells could be collected by MACS fromeach Buffy Coat. However, in future clinical applications, startingsamples will be Leukapheresis products that contain larger numbers ofcells and are collected by an Apheresis machine. In previous studies,unstimulated leukapheresis products collected from leukemia patientscontained on average 13.4×10⁹ (range 4.4-20.6×10⁹) peripheral bloodcells, of which around 160 million TCRγδ⁺ T cells (range 1.0-3.0×10⁸)were obtained after MACS (Wilhelm, M., et al., Successful adoptivetransfer and in vivo expansion of haploidentical gammadelta T cells. JTransl Med, 2014. 12: p. 45.). This represents, on average, about 9times more initial cells than what was obtained with Buffy Coats.Consequently, the average estimated number of cells that would begenerated in the same culture system if leukapheresis products were usedas starting sample is about 10.2×10⁹ cells (range: 3.9×10⁹ 14.4×10⁹cells).

Table 14 shows reagents and materials used to produce pharmaceuticalgrade TCRγδ⁺ T cells.

EXAMPLES

Optimization of the Ex-Vivo Expansion of Human Vδ1⁺ TCRγδ⁺ T Cells

The inventors performed a series of experiments aiming to improve theexpansion and purity levels of in vitro cultured Vδ2⁻ γδ T cells. Sincethere was no commercially available antibody against the Vδ3⁺ chain ofthe TCR, an anti-TCRVδ1 mAb was used to identify Vδ1⁺ T cells in cellsamples, during the culture optimization stage. TCRγδ⁺ T PBLs from apanel of healthy donors were isolated by MACS and tested for theirreactivity to in vitro stimulation with IL-2 and PHA (i.e., detectablechanges in cell activation and proliferation). One donor with reactiveVδ1⁺ PBLs was selected to provide blood samples for the rest of theoptimization study. The preference for a fixed healthy donor wasimportant, since a more reliable comparison could be performed betweenresults obtained in different experiments. The selected donor had anormal (but high) percentage of TCRγδ⁺ T cells in the peripheral blood(10%-12% of total PBLs), although a very low percentage of Vδ1⁺ PBLs(0.3% of total PBLs, or 3.0% of total TCRγδ⁺ T PBLs; FIG. 1). He wasconsidered a suitable donor to use in these experiments since everyminor improvement in the final number and purity levels of cultured Vδ1⁺T-cells could be readily detected by flow cytometry.

The single-step MACS protocol used to isolate TCRγδ⁺ T cells from PBMCswas very efficient, generating highly pure cell populations (FIG. 1).However, one critical reagent (i.e., the human anti-TCRγδ mAb conjugatedwith magnetic microbeads, from Miltenyi Biotec) is not currentlyapproved for clinical applications. The clinical-grade production,validation and regulatory approval of such reagent can take many years,and this problem will prevent any immediate use of the describedprotocol in clinical applications. A different, but equivalent methodfor isolating TCRγδ⁺ T cells was then developed and adopted. The processconsisted of two steps of magnetic cell separation, as described in FIG.2. The final purity levels of the obtained TCRγδ⁺ T cells were lowerthan with the previous (single-step) method, but still acceptable forcell culture. Importantly, this new cell isolation method used onlyreagents already approved for clinical applications (manufactured byMiltenyi Biotec GmbH, Germany).

The inventors then tested multiple combinations of clinical-gradeagonist antibodies and cytokines for their capacity to expand anddifferentiate (over 2-3 weeks) Vδ1⁺ T cells from the peripheral blood.MACS-sorted TCRγδ⁺ PBLs collected from the previously selected healthydonor were incubated in culture medium for 2-3 weeks in 96-well plates,at 37° C. and 5% CO₂, in the presence of 58 different T/NK cellactivating molecules (Table 1). These included 13 different TCRagonists, 23 different co-receptor agonists, and 22 different cytokines,which were tested in 2.488 different combinations and concentrations.Antibodies were used in both soluble and plastic-bound presentations.Cytokines were tested at a concentration range from 0.1 ng/ml to 1000ng/ml; TCR agonists were used at 0.1 ng/ml to 40 μg/ml, and co-receptoragonists were used at final concentration 0.5 μg/ml to 80 μg/ml.

Several sequential cell isolation and cell culture expansion experimentswere performed from the same donor; each experiment testing the effectof about 100-400 different combinations of activating molecules. Theoptimization started from the basic, non-optimized cocktail (i.e.,culturing TCRγδ⁺ T cells in the presence of IL-2 and PHA). Fresh mediumcontaining the same chosen cocktail of activating molecules was addedevery 5 days. After 14 days, cells were collected and their phenotypewas analyzed by flow cytometry. The best culture condition of eachexperiment was identified (for the highest fold expansion of Vδ1⁺ Tcells), and selected for further optimization, combined to all availablereagents, tested at various concentrations. Fold expansion and puritylevels of Vδ1⁺ T cells gradually increased during the optimizationstage, after each superior culture condition was obtained.

Results of experiments 1-4 are summarized in Table 2. Experiment n^(o).1 confirmed previous observations that IL-4 is a key growth factor inpromoting Vδ1⁺ T cell proliferation and enrichment in culture.^(27, 42)In this experiment, the inventors tested the activity of 22 differentcytokines on cultured TCRγδ⁺ T cells, in the presence of a T cellmitogen and IL-2. Clearly, IL-4 was unique in the ability to induce astrong enrichment and expansion of these cells. In contrast, the use ofincreasing concentrations of IL-2, or the combination of IL-2 withdifferent T cell mitogens, did not produce an equivalent effect, mostprobably because of increased activation-induced-cell-death (AICD) ofcultured cells (conditions 2-3; Table 2).

TABLE 1 TCR Monoclonal anti-human TCR Vδ1 mAb (Clone TS8.2); purifiedThermo Fisher Sci. agonists antibodies anti-human TCR δTCS-1 mAb (CloneTS-1); purified Thermo Fisher Sci. (soluble and anti-human TCR PAN γδmAb (Clone IMMU510); purified Beckman Coulter plate-bound) anti-humanCD3 mAb (Clone OKT3); purified BioXcell/ Biolegend Plant lectins Lectinfrom Phaseolus vulgaris (red bean; PHA-P), pur. Sigma-Aldrich, Co.(soluble) Concanavalin A (from Canavalia ensiformis; Con-A), pur. Lectinfrom Phytolacca americana; purified Lectin from Triticum vulgaris(wheat); purified Lectin from Lens culinaris (lentil); purified Lectinfrom Glycine max (soybean); purified Lectin from Maackia amurensis;purified Lectin from Pisum sativum (pea); purified Lectin from Sambucusnigra (elder); purified Co- Monoclonal anti-human CD2 mAb (Clone S5.2);purified BD Biosciences receptor antibodies anti-human CD6 mAb (CloneUMCD6/3F7B5); purified Ancell Corporation agonists (soluble andanti-human CD9 mAb (Clone MEM-61); purified Exbio Praha, a.s.plate-bound) anti-human CD28 mAb (Clone CD28.2); purified Biolegendanti-human CD43 mAb (Clone MEM-59); purified Exbio Praha, a.s.anti-human CD94 mAb (Clone HP-3B1); purified Santa Cruz Biotechanti-human CD160 mAb (Clone CL1-R2); purified Novus Biologicalsanti-human SLAM mAb [Clone A12(7D4)]; purified Biolegend anti-humanNKG2D mAb (Clone 1D11); purified Exbio Praha, a.s. anti-human 2B4 mAb(Clone C1.7); purified Biolegend anti-human HLA-A, B, C mAb (CloneW6/32); purified Biolegend anti-human ICAM-3 mAb (Clone MEM-171);purified Exbio Praha, a.s. anti-human ICOS mAb (Clone C398.4A); purifiedBiolegend Recombinant Human SECTM-1/Fc Chimera (CD7 ligand) R&D Systemsproteins Human CD26 (Dipeptidyl Peptidase IV) Sigma-Aldrich, Co.(soluble) Human CD27L (CD27 ligand); PeproTech, inc. Human CD30L (CD30ligand); PeproTech, inc. Human CD40L (CD40 ligand); PeproTech, inc.Human OX40L (OX40 ligand); PeproTech, inc. Human 4-1BBL (4-1BB ligand)PeproTech, inc. Human ICAM-1 PeproTech, inc. Human FibronectinSigma-Aldrich, Co. Human Hydrocortisone Sigma-Aldrich, Co. CytokinesRecombinant Human IFN-γ (Interferon-γ); PeproTech, inc. proteins HumanTGF-β (Transforming growth factor beta); PeproTech, inc. (soluble) HumanIL-1-β (interleukin-1β); PeproTech, inc. Human IL-2 (interleukin-2);PeproTech, inc. Human IL-3 (interleukin-3); PeproTech, inc. Human IL-4(interleukin-4); PeproTech, inc. Human IL-6 (interleukin-6); BiolegendHuman IL-7 (interleukin-7); PeproTech, inc. Human IL-9 (interleukin-9);PeproTech, inc. Human IL-10 (interleukin-10); PeproTech, inc. HumanIL-12 (interleukin-12); PeproTech, inc. Human IL-13 (interleukin-13);PeproTech, inc. Human IL-15 (interleukin-15); PeproTech, inc Human IL-18(interleukin-18); Southern Biotech Human IL-21 (interleukin-21);PeproTech, inc. Human IL-22 (interleukin-22); PeproTech, inc. HumanIL-23 (interleukin-23); PeproTech, inc. Human IL-27 (interleukin-27);Biolegend Human IL-31 (interleukin-31); PeproTech, inc. Human IL-33(interleukin-33); PeproTech, inc. Human GM-CSF (Granul.-macroph. col.stimul. factor); PeproTech, inc. Human FLT3-L (FMS-like tyrosine kinase3 ligand); PeproTech, inc.

TABLE 2 Total Fold Live Vδ1⁺ increase Cond. Condition: cells T cells ofVδ1⁺ Exp. No (cultured 1 million cells/ml for 14 days in 96-well plates)(%) (%) T cells 1 1 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 68.931.6 77 2 500 ng/ml IL-2 + 1 μg/ml PHA 63.3 10.5  4 3 20 ng/ml IL-2 + 1μg/ml PHA (control) 68.8 1.90  1 2 1 20 ng/ml IL-2 + 1 μg/ml α-CD3 mAb +20 ng/ml IL-4 90.0 51.7 75 2 20 ng/ml IL-2 + 1 μg/ml α-Vδ1 TCR mAb + 20ng/ml IL-4 85.2 55.9 69 3 5 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-484.0 61.9 62 4 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 (previousbest) 72.0 45.3 27 5 100 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 71.355.7 22 6 300 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 71.3 57.0 21 3 15 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 91.7 61.4 138  2 5ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 81.4 59.4 124  3 20 ng/mlIL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best) 84.2 45.4 105  4 5ng/ml IL-15 + 1 μg/ml PHA + 20 ng/ml IL-4 68.0 76.2 21 5 5 ng/ml IL-2 +1 μg/ml PHA + 20 ng/ml IL-4 60.1 69.1 19 6 20 ng/ml IL-15 + 1 μg/mlPHA + 20 ng/ml IL-4 68.6 69.9 13 7 20 ng/ml IL-2 + 1 μg/ml PHA + 20ng/ml IL-4 62.9 67.7 11 4 1 20 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20ng/ml IL-4 87.1 79.5 1 349   2 3 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20ng/ml IL-4 85.5 67.4 1 014   3 2 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20ng/ml IL-4 87.9 81.6 909  4 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 85.9 67.8804  5 5 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best)78.9 69.4 624 

Results of experiment n^(o). 2 demonstrated that IL-2 (when in thepresence of IL-4 and PHA) must be used at low concentrations, forincreased proliferation and enrichment of Vδ1⁺ T cells in culture(conditions 3-7, Table 2). This effect was not observed in the previousexperiment (exp. n^(o) 1), in which fold expansion was higher in thepresence of PHA and high levels of IL-2, in the absence of IL-4.Furthermore, the anti-CD3 mAb was shown to be the most effective mitogenpromoting survival and proliferation of Vδ1⁺ T cells in culture (seecondition 1 versus condition 2, Table 2).

Results of experiment n^(o) 0.3 (Table 2) confirmed previousobservations and showed that even lower (than previously used)concentration levels of IL-2 (in the presence of IL-4 and a T cellmitogen), promoted even higher Vδ1⁺ T cell proliferation, survival andenrichment in culture. Also, when used at the same concentration, IL-15was more effective than IL-2 in promoting Vδ1⁺ T cell survival,enrichment and proliferation. Again, the anti-CD3 mAb was shown to bethe most effective mitogen of our tests.

Results of experiment n^(o) 4 further indicated that the presence ofhigh levels of IL-15 in the culture medium was detrimental for theproliferation of Vδ1⁺ T cells. Indeed, even lower (than previously used)concentration levels of IL-15 promoted even higher expansion levels ofVδ1⁺ T cells, when in the presence of IL-4 and α-CD3 (Conditions 3-5,Table 2). Finally, in a totally unexpected finding, the replacement ofIL-15 by INF-γ (i.e., the absence of IL-15 and the presence of IFN-γ inthe culture medium), was shown to generate increased expansion levels ofcultured Vδ1⁺ T cells.

Although many different concentrations and combinations of IL-2, IL-7and IL-15 were tested in parallel, IFN-γ was consistently found to be amuch more effective reagent for promoting the selective expansion ofVδ1⁺ T cells in culture (when used in the presence of IL-4 and a T cellmitogen, such as anti-CD3 mAb; FIG. 3). The higher performance of IFN-γin these cultures was independent of the concentrations of IL-4 andanti-CD3 mAb used (FIG. 3A-C and FIG. 4). Importantly, the use of IFN-γ(but not of IL-2, IL-15 or IL-7), induced a drastic increase in theenrichment and expansion levels of Vδ1⁺ T cells in bulk cultures of CD3⁺T cells (FIG. 3D). In these cultures, contaminating TCRαβ⁺ T cellspresent in the starting samples responded to and proliferated in thepresence of IL-2, IL-15 and IL-7, but not in the presence of IFN-γ, thatseems to be a more selective activator of Vδ1⁺ T cells. Theseexperiments showed that the specific (and not previously described)combination of IFN-γ with IL-4 and a T cell mitogen, in the absence ofIL-2, IL-7 and IL-15 for the production of Vδ1⁺ T cells has uniqueadvantages that can be used in a variety of novel therapeutic andcommercial applications.

IFN-γ is a dimerized soluble cytokine and is the only member of the typeII class of interferons.⁵⁰ It is structurally and functionally differentfrom common γ-chain cytokines such as IL-2, IL-4, IL-7 or IL-15 and isserologically distinct from Type I interferons: it is acid-labile, whilethe type I variants are acid-stable.

Although we have obtained a new and improved method for expanding andenriching populations of Vδ1⁺ T cells in culture, we also sought toanalyze the anti-tumor function of the generated cells, in vitro. Wefound that the presence of IL-4 in the culture medium strongly inhibitedor decreased the expression of activating receptors such as NaturalCytotoxicity Receptors (NCRs, namely NKp30 and NKp44), and NKG2D onexpanded Vδ1⁺ T-cells (Table 3).

Activating Natural Killer (NK) receptors (such as NKp30, NKp44 andNKG2D) are known to play a critical role in the anti-tumor andanti-viral function of Vδ1⁺ T cells, through binding to their molecularligands expressed on the surface of tumor or infected cells.Receptor-ligand binding triggers the production and release of granzymesand perforines by Vδ1⁺ T cells, leading to the death of target cells.²⁹In our study, we found that the presence of IL-4 in the culture mediuminduced a decrease in the levels of activating NK receptors located atthe surface of cultured TCRγδ⁺ T cells, thereby decreasing theircytotoxic function against MOLT-4 leukemia cells (Table 3). Of note, theinhibition induced by IL-4 seemed to affect all TCRγδ⁺ T cell subsetspresent in the final cellular product, including both Vδ1⁻ and Vδ1⁺TCRγδ⁺ T cells (Table 3).

TABLE 3 Fold Vδ1⁺ Vδ1⁺ Vδ1⁺ Dead Culture Culture Live Vδ1⁺ T increaseNKp30⁺ NKp44⁺ NKG2D⁺ tumor condition condition cells cells of Vδ1⁺ Tcells T cells T cells target Condition N^(o) (days 1-14) (days 15-21)(%) (%) T cells (%) (%) (%) cells (%) 1 100 ng/ml IL-15 61.4 50.3   10845.0 38.0 99.8 62.6 1 μg/ml a-CD3 mAb (control) 2 100 ng/ml IL-4 81.092.6 8 064 0.9 0.2 48.2 14.0 70 ng/ml IFN-γ 1 μg/ml a-CD3 mAb 3 100ng/ml IL-4 83.1 83.1 2 185 0.1 0.3 46.4 17.5 1 μg/ml a-CD3 mAb 4 100ng/ml IL-4 68.4 92.8 1 263 1.6 1.1 38.4 13.3 1 μg/ml a-TCRVδ1 mAb 5 100ng/ml IL-4 69.6 73.8   464 1.5 2.2 58.7 9.0 1 μg/ml PHA 6 100 ng/ml IL-4100 ng/ml IL-15 87.3 85.9 24 152  30.1 14.7 98.9 68.5 70 ng/ml IFN-γ 1μg/ml a-CD3 mAb 7 1 μg/ml a-CD3 mAb 100 ng/ml IL-2 80.1 88.1 16 374 29.0 18.2 99.4 70.2 1 μg/ml a-CD3 mAb 8 100 ng/ml IL-7 78.3 85.4 18 366 15.6 15.4 99.2 67.6 1 μg/ml a-CD3 mAb 9 100 ng/ml IL-4 100 ng/ml IL-1583.1 80.6 9 636 19.5 17.7 99.0 64.6 1 μg/ml a-CD3 mAb 1 μg/ml a-CD3 mAb10 100 ng/ml IL-2 84.0 85.5 7 747 23.0 12.9 98.4 54.4 1 μg/ml a-CD3 mAb11 100 ng/ml IL-7 76.5 83.0 10 567  10.1 4.4 99.2 50.6 1 μg/ml a-CD3 mAb12 100 ng/ml IL-4 100 ng/ml IL-15 69.4 72.6 5 564 23.2 6.6 95.4 54.5 1μg/ml a-Vδ1 mAb 1 μg/ml a-Vδ1 mAb 13 100 ng/ml IL-4 100 ng/ml IL-15 67.865.6 2 454 17.6 13.4 95.6 46.0 1 μg/ml PHA 1 μg/ml PHA

This was in line with a recent study showing that IL-4 promotes thegeneration of regulatory Vδ1⁺ T cells via IL-10 production. IL-4-treatedVδ1⁺ T cells secreted significantly less IFN-γ and more IL-10 relativeto Vδ2⁺ T cells. Furthermore, Vδ1⁺ T cells showed relatively low levelsof NKG2D expression in the presence of IL-4, suggesting that Vδ1⁺ Tcells weaken the TCRγδ⁺ T cell-mediated anti-tumor immune response.⁴²

Since we were looking for novel culture methods with the objective ofimproving the anti-tumor effector functions of Vδ1⁺ T cells, weattempted to recover the expression of activating NK receptors onIL-4-treated cells, also recovering the cytotoxic phenotype of thesecells, something that was never tried before.

TCRγδ⁺ T cells were cultured in a two-step protocol. The first stepconsisted of treating cells in culture medium in the presence of a Tcell mitogen (such as anti-CD3 mAb or PHA) and IL-4, and in the absenceof IL-2, IL-15 and IL-7, to promote the selective expansion of Vδ1⁺T-cells. The second culture step consisted of treating cells in aculture medium in the absence of IL-4, and in the presence of a T cellmitogen and IL-2, or IL-7 or IL-15, to promote cell differentiation, andNKR expression (Table 3 and Table 4).

Conditions 1-5 (Table 3) confirmed previous results, showing that Vδ1⁺ Tcells cultured in the presence of IL-4 could expand in culture severalthousand fold, but could not differentiate, becoming inefficient killersof tumor cells. In contrast, as seen in conditions 6-13, when cells weresubcultured in a second culture medium in the absence of IL-4 and in thepresence of a T cell mitogen and IL-2, or IL-7, or IL-15, the ability toeliminate tumor cells increased radically. The presence of each one ofthese three cytokines (IL-2, IL-7 and IL-15), alone or in combination,was able to revert the phenotype of cultured Vδ1⁺ T cells and thus canbe used for this purpose.

Results of experiments n^(o). 6 and 7 (Table 4) showed that 2-stepprotocols and even 3-step protocols could be more efficiently used tostimulate the proliferation and differentiation of Vδ1⁺ T-cells. In thecase of 3-step protocols, where cells were cultured in 3 differentculture media (see for example condition 2 of experiment n^(o) 7 ofTable 4), it was very important to separate the IL-4 containing mediumfrom medium containing IL-2 or IL-7 or IL-15. From these results itcould also be concluded that a fraction of old culture medium should beremoved during each subculture step for improved cell expansion; andthat in the second culture medium, IL-15 is slightly more efficient thanIL-2 in promoting Vδ1⁺ T-cell proliferation.

Additional experiments using 3-step and 4-step culture protocols furtherdemonstrated that other growth factors can be added to the first and/orsecond culture medium (Table 3 and Table 4) for increased expansionlevels of Vδ1⁺ T cells and expression of NK receptors on these cells.INF-γ, IL-21 and IL-1β were identified as efficient inducers of Vδ1⁺ Tcell expansion and survival (Table 5). These growth factors could beused in the first or in the second culture media.

Finally, the addition of a soluble ligand of the CD27 receptor, or asoluble ligand of the CD7 receptor or a soluble ligand of SLAM receptorresulted in enhanced expansion of Vδ1⁺ T cells (Conditions 3-δ of Table5). CD27 receptor is typically required for the generation and long-termmaintenance of T cell immunity. It binds to its ligand CD70, and plays akey role in regulating B-cell activation and immunoglobulin synthesis.CD7 receptor is a member of the immunoglobulin superfamily. This proteinis found on thymocytes and mature T cells. It plays an essential role inT-cell interactions and also in T-cell/B-cell interaction during earlylymphoid development. SLAM receptor is a member of the signalinglymphocytic activation molecule family of immunomodulatory receptors.

TABLE 4 Fold Vδ1⁺ Vδ1⁺ increase NKp30⁺ Cond. Condition: T cells of Vδ1⁺T cells Exp. No (cultured 5 × 10⁵ million cells/ml for 15 days in96-well plates): (%) T cells (%) 6 1 Days 0-5: 100 ng/ml IL-4 + 1 μg/mlα-CD3 + 70 ng/ml IFN-γ 72.4 7 635 39.4 Days 6-15: 100 ng/ml IL-15 + 2μg/ml α-CD3 2 Days 0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ60.1 5 100 37.9 Days 6-15: 100 ng/ml IL-7 + 2 μg/ml α-CD3 3 Days 0-5:100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 68.2 4 135 36.5 Days6-15: 100 ng/ml IL-2 + 2 μg/ml α-CD3 7 1 Days 0-5: 100 ng/ml IL-4 + 1μg/ml α-CD3 + 70 ng/ml IFN-γ 65.0 4 468 45.4 Days 6-10: 100 ng/mlIL-15 + 2 μg/ml α-CD3 Days 11-15: remove medium, 100 ng/ml IL-15 + 2μg/ml α-CD3 2 Days 0-5: 30 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ80.5 3 987 36.0 Days 6-10: 100 ng/ml IL-15 + 1 μg/ml α-CD3 + 2 ng/mlIL-21 Days 11-15: 100 ng/ml IL-15 + 1 μg/ml α-CD3 + 5 ng/ml IL-21 3 Days0-5: 100 ng/ml IL-4 + 1 μg/ml α-CD3 + 70 ng/ml IFN-γ 64.0 3 683 41.0Days 6-10: remove medium, 100 ng/ml IL-15 + 2 μg/ml α-CD3 Days 11-15:100 ng/ml IL-15 + 2 μg/ml α-CD3

Of note, several different culture media were tested (FIG. 5). Thesetests have shown that the present invention works very well withdifferent culture media, including commercially available, serum free,clinical-grade media produced by different manufacturers, and suitablefor clinical applications.

TABLE 5 Fold Vδ1⁺ Culture Culture Culture Culture Vδ1⁺ T increase NKp30⁺Condition condition condition condition condition cells of Vδ1⁺ T cellsnumber: (days 1-6) (days 7-11) (days 12-16) (days 17-21) (%) T cells (%)1 100 ng/ml IL-4 70 ng/ml IFN-γ 75.0 61 417 54.1 70 ng/ml IFN-γ 2 μg/mlα-CD3 mAb 70 ng/ml α-CD3 mAb 100 ng/ml IL-15 70 ng/ml IL-21 15 ng/mlIL-1β 2 100 ng/ml IL-4 70 ng/ml IFN-γ 80.1 37 457 38.5 70 ng/ml IFN-γ 2μg/ml α-CD3 mAb 70 ng/ml α-CD3 mAb 100 ng/ml IL-15 70 ng/ml IL-21 3 100ng/ml IL-4 1 μg/ml α-CD3 mAb 72.4 10 535 22.5 70 ng/ml IFN-γ 100 ng/mlIL-15 70 ng/ml α-CD3 mAb 1 μg/ml sCD27L 4 100 ng/ml IL-4 69.6  9 56625.4 70 ng/ml IFN-γ 70 ng/ml α-CD3 mAb 1 μg/ml α-SLAM mAb 5 100 ng/mlIL-4 70.7  7 764 24.5 70 ng/ml IFN-γ 70 ng/ml α-CD3 mAb 1 μg/ml sCD7L 6100 ng/ml IL-4 72.8  5 594 21.4 70 ng/ml IFN-γ 70 ng/ml α-CD3 mAb

In Vitro Characterization of Large-Scale Expanded TCRγδ⁺ T Cells

Having established an effective protocol for the isolation and expansionof Vδ1⁺ T cells in culture, we sought to test it with blood samplescollected from a larger number of healthy donors and also from cancerpatients. This was necessary to test the robustness and generalapplicability of the new culture method. Moreover, instead of plasticplates or flasks, cells were cultured in closed, large-scale,gas-permeable cell bags developed for clinical applications.

The adopted two-step method of magnetic-associated cell sorting (MACS)produced viable cell populations enriched in TCRγδ⁺ T cells from 8different donors (Table 6). Vδ1⁺ TCRγδ⁺ T cells comprised only about 1%to 44% of the total viable cells initially present after MACS. However,within 11-21 days of treatment following the optimized 2-step culturemethod and in the presence of the described cocktail of cytokines and Tcell mitogen, Vδ1⁺ T cells became the dominant cell subset in culture,varying between 60-80% of total cells between donors (FIG. 6). Of note,a very reproducible expansion was achieved and the composition of thefinal cellular product was remarkably similar across multiple donors(FIG. 6, Table 7). Importantly non-Vδ1⁺ TCRγδ⁺ T cells in the finalproducts were found to be mostly Vδ1⁻ Vδ2⁻ TCRγδ⁺ T cells (whichcomprised around 17%-37% of total cells). These cells probably consistedof Vδ3⁺ TCRγδ⁺ T cells, since this is the third most abundant TCRγδ⁺ Tcell subset in the peripheral blood. It was possible to calculate thepercentage of Vδ1⁻ Vδ2⁻ TCRγδ⁺ T cells in the final cellular products bysubtracting the percentage of Vδ1⁺ T cells and Vδ2⁺ T cells from thepercentage of total TCRγδ⁺ T cells (Table 7). Fold expansions in theseplastic bags were, as expected, of lower magnitude than those fromplates, but still generated relevant numbers for clinical translation.

The expression of activating Natural Cytotoxicity Receptors (NCRs;including NKp30 and NKp44), and NKG2D was robustly induced in long-termcultured Vδ1⁺ T cells, of all tested donors (Table 8 and FIG. 7). Theobtained TCRγδ⁺ T cells (which included both Vδ1⁺ and Vδ1⁻ Vδ2⁻ cellsubsets) were highly cytotoxic against CLL cells (both MEC-1 cell lineand primary CLL patient samples), but did not target healthy autologousPBMCs (FIG. 8A and FIG. 8C). Preliminary experiments with the use ofblocking antibodies against TCRγδ⁺ T cell activating receptors showedthat anti-tumor cytotoxicity was partially reliant on NKG2D and NKp30receptors (FIG. 8B). Furthermore, expanded and differentiated TCRγδ⁺ Tcells produced high levels of the pro-inflammatory cytokine IFN-γ (FIG.9).

Finally, Vδ2⁻ TCRγδ⁺ T cells could be efficiently isolated and expandedfrom the PBLs of elderly CLL patients with very high tumour burden(Table 9 and Table 10 and FIG. 10A), and displayed potent anti-tumourand anti-viral activities (FIG. 10B). Of note, contaminating autologousleukemic B cells were eliminated during the in vitro culture of TCRγδ⁺ Tcells (Table 10).

This collection of data fully demonstrates the unique ability of theinvention to generate functional Vδ2⁻ γδ T cells (namely from cancerpatients) for autologous or allogeneic adoptive cell therapy. The methodis robust enough to enrich (>60%) and expand (up to 2,000-fold) Vδ1⁺ Tcells from highly unpurified samples obtained from CLL patients,differentiating them into NKR-expressing and highly cytotoxic TCRγδ⁺ Tcells.

Importantly, preliminary tests also demonstrated in vitro reactivity ofcultured TCRγδ⁺ T cells against tumor cells of other tissue origins(FIG. 11), suggesting that expanded and differentiated Vδ2⁻ γδ T cellscan also be used for treating these conditions.

TABLE 6 Cell lineage: B cells TCRαβ⁺ TCRγδ⁺ T Vδ1⁺ T Vδ2⁺ T Total cell(CD19⁺ NK cells T cells T cells cells cells cells viability CD20⁺ (CD56⁺(CD3⁺ (TCRαβ⁺ (TCRγδ⁺ (TCRVδ1⁺ (TCRVδ2⁺ (Trypan Donor cells) CD3⁻ cells)cells) CD3⁺ cells) CD3⁺ cells) CD3⁺ cells) CD3⁺ cells) Blue⁻ cells) A24.6 9.92 43.5 0.01 41.9 13.5 23.6 79.4 B 6.69 0.07 75.0 0.71 63.2 21.630.0 80.1 C 1.36 0.36 95.3 0.37 94.5 1.25 92.6 95.2 D 13.9 6.79 41.20.76 40.2 18.3 21.5 89.1 E 17.0 1.84 65.8 2.80 59.6 17.7 40.9 89.9 F0.28 8.14 91.4 0.81 90.0 2.05 86.1 93.7 G 7.32 0.25 87.5 0.58 81.8 24.045.0 84.0 H 3.51 0.10 88.8 0.80 84.8 44.0 27.0 86.0

TABLE 7 Cell lineage: B cells TCRαβ⁺ T TCRγδ⁺ T Vδ1⁺ T Vδ2⁺ T Total cell(CD19⁺ NK cells T cells cells cells cells cells viability CD20⁺ (CD56⁺CD3⁻ (CD3⁺ (TCRαβ⁺ (TCRγδ⁺ (TCRVδ1⁺ (TCRVδ2⁺ (Trypan Blue⁻ Donor cells)cells) cells) CD3⁺ cells) CD3⁺ cells) CD3⁺ cells) CD3⁺ cells) cells) A 00.50 99.5 0.01 99.3 82.6 3.9 89.0 B 0 0.02 99.7 0.06 99.5 80.8 3.7 93.3C 0 0.50 96.3 0.03 92.8 69.9 4.2 90.3 D 0 0.03 99.6 0.02 99.1 62.2 2.394.5 E 0 0.11 99.5 0.01 99.2 63.3 3.3 95.9 F 0 0 99.9 0.02 98.0 73.3 4.393.2 G 0 0.10 99.4 0.01 98.4 71.7 3.5 90.0 H 0 0.40 97.5 0 98.2 72.0 1.689.0

TABLE 8 Activating Donor receptor Day 0 Day 16 Day 21 A NKp30 0.51 66.865.0 NKp44 0.30 18.3 23.3 NKG2D 46.0 96.5 98.0 B NKp30 0.56 71.6 68.0NKp44 0 37.2 38.7 NKG2D 55.0 90.7 95.1

TABLE 9 Cell lineage: B cells TCRαβ⁺ T TCRγδ⁺ T Vδ1⁺ T (CD19⁺ NK cells Tcells cells cells cells Cell viability CD20⁺ (CD56⁺ (CD3⁺ (TCRαβ⁺(TCRγδ⁺ CD3⁺ (TCRVδ1⁺ (Trypan Blue⁻ Donor cells) CD3⁻ cells) cells) CD3⁺cells) cells) CD3⁺ cells) cells) Before MACS (Day 0) CLL-1 63.4 1.2230.4 27.5 0.66 0.22 92.0 CLL-2 85.7 0.92 8.35 6.97 0.43 0.03 90.0 CLL-390.4 0.15 3.74 3.31 0.35 1.9 × 10⁻³ 87.0 After MACS (Day 0) CLL-1 38.00.72 37.2 0.19 7.32 4.00 88.0 CLL-2 35.4 0.26 61.3 0.05 36.7 1.70 83.0CLL-3 57.0 0.45 39.5 0.02 10.4 0.28 80.0

TABLE 10 Cell phenotype after in vitro culture (Day 21) Cell lineage: Bcells TCRαβ⁺ T TCRγδ⁺ T Vδ1⁺ T NKp30⁺ NKG2D⁺ (CD19⁺ NK cells T cellscells cells cells Vδ1⁺ T Vδ1⁺ T CD20⁺ (CD56⁺ (CD3⁺ (TCRαβ⁺ (TCRγδ⁺(TCRVδ1⁺ cells cells Donor cells) CD3⁻ cells) cells) CD3⁺ cells) CD3⁺cells) CD3⁺ cells) (pre-gated) (pre-gated) CLL-1 0.04 0.11 96.8 0.0894.1 60.1 23.0 95.6 CLL-2 0.07 0.01 99.5 0.02 97.4 80.0 11.0 98.9 CLL-30.05 0.01 99.8 0.01 99.6 70.1 13.4 97.2

In Vivo Studies of Expanded TCRγδ⁺ T Cells

Having successfully developed a method to generate large numbers offunctional TCRγδ⁺ T cells, which we called “DOT-cells”, we nextinvestigated their homing and anti-leukaemia activity in vivo. We tookadvantage of a xenograft model of human CLL previously shown toreproduce several aspects of the disease and used to test the efficacyof other cellular therapies, including CAR-T cells.^(51, 52) The modelrelies on the adoptive transfer of CLL/SLL-derived MEC-1 cells intoBalb/c Rag^(−/−) γc^(−/−) (BRG) animals, which lack all lymphocytes andthus do not immediately reject the human cells. However, some myeloidlineage-mediated rejection of human xenografts still occurs. Thisrejection varies in its magnitude according to the mouse strain used,due to different alleles encoding for SIRP-α⁵³. We have thus furtheradapted the model in order to characterize TCRγδ⁺ T cells at late timepoints after transfer, using NOD-SCID γc^(−/−) (NSG) animals as hosts.Indeed, upon transfer into tumour-bearing NSG hosts we were able torecover TCRγδ⁺ T cells in all tissues analysed, 30 days after transfer,with a strong enrichment for CD3±Vδ1⁺ T cells (FIG. 12). Importantly, wedetected the expression of NKp30 and NKG2D in the recovered TCRγδ⁺ Tcells, demonstrating that they stably preserve their characteristics invivo. Of note, upon transfer into BRG animals we could not recoverTCRγδ⁺ T cells at such late time points, but we observed them in bothlung and liver at 72 hours after transfer (FIG. 12). These experimentsconfirm the better fitness of human xenografted cells in NSG animals,while allowing us to have two different models to test the anti-tumourproperties of the expanded Vδ2⁻ γδ T cells in vivo.

In order to dynamically follow tumour growth using bioluminescence, wetransduced MEC-1 cells with firefly luciferase-GFP, and transferred 10⁷MEC-1 cells sub-cutaneously into BRG animals. After 7 days we injectedluciferin i.p. to determine tumour load as a function of luminescence,before ascribing treatment (or PBS control) to the animals. We performedtwo transfers of TCRγδ⁺ T cells within 5 days. We then measured tumoursize as a function of time using a Caliper; importantly, we detected aclear reduction in primary tumour size in treated animals when comparedto controls (FIG. 13). This reduction was significant 9 days after thesecond transfer of TCRγδ⁺ T cells. This result demonstrated that TCRγδ⁺T cells are effective in vivo, even if a more extensive characterizationof their anti-tumour properties was precluded by the short half-life ofthe human cells in BRG hosts. To overcome this limitation, we nextperformed a similar experiment using NSG animals as hosts.

Tumour progression growth was faster in NSG hosts, which seeminglyprevented TCRγδ⁺ T cells from interfering with primary tumour growth(FIG. 14). However, in this model the tumour disseminates to variousorgans at late time points, and we found that TCRγδ⁺ T cells werestrikingly capable of limiting tumour spread as documented by flowcytometry and histological analysis of an array of tissues (FIG. 14B-D).This included dissemination sites such as the bone marrow and the liver.The data obtained in the two xenograft models collectively demonstratethe in vivo efficacy of TCRγδ⁺ T cells to reduce primary tumour size (inBRG hosts; FIG. 13) and to control of tumour dissemination to targetorgans (in NSG hosts; FIG. 14).

Examination of the TCRγδ⁺ T cell progeny at the end of the experiment inthe NSG model confirmed robust infiltration into the tumour tissue (FIG.15A); and the stable expression of NKp30 and NKG2D on TCRγδ⁺ T cells(FIG. 15B). Interestingly, we detected high expression of the earlyactivation marker CD69 specifically in tumour-infiltrating TCRγδ⁺ Tcells, suggesting optimal activation of TCRγδ⁺ T cells within the tumour(FIG. 15B). Importantly, we did not observe any treatment-associatedtoxicity upon histological analyses (of multiple organs); or biochemicalanalysis of blood collected at time of necropsy (FIG. 16).

Collectively, these in vivo data provide great confidence in the safetyand efficacy of the generated TCRγδ⁺ T cells for CLL treatment, thusinspiring their clinical application.

In conclusion, we have developed a new and robust (highly reproducible)clinical-grade method, devoid of feeder cells, for selective andlarge-scale expansion and differentiation of cytotoxic Vδ2⁻ γδ T cells;and tested their therapeutic potential in pre-clinical models of chroniclymphocytic leukemia (CLL). Our cellular product, named DOT-cells, doesnot involve any genetic manipulation; and specifically targets leukemicbut not healthy cells in vitro; and prevents wide-scale tumordissemination to peripheral organs in vivo, without any signs of healthytissue damage. Our results provide new means and the proof-of-principlefor clinical application of DOT-cells in adoptive immunotherapy ofcancer.

Supplementary Data

The following section discloses additional data generated with the useof the previously described invention. The data contained hereinconfirmed previous results and expanded on previous observations andshould be used as supporting information for a better understanding ofthe subject matter.

As previously explained, the combination of interleukin-2 (IL-2) andinterleukin-4 (IL-4) has been used with some success to expand Vδ1⁺ Tcells in vitro. However, we found that the presence of IL-4 in theculture medium induced a strong downregulation of natural killer (NK)activating receptors (such as NKG2D, NKp30 and NKp44) on cultured TCRγδ⁺T cells, weakening their anti-tumor responses.

Our previous results of experiments 1-4 are presented here again in moredetail (see Table 11). Additional results obtained in parallel cultureconditions (marked with an asterisk) are shown, for a more completeunderstanding of the results. It is also disclosed herein the percentageof NKp30±Vδ1⁺ T cells observed after cell culture with each condition.The observed downregulation of expression of NKp30 on cultured cellsfurther confirmed that the potent inhibitory effects of IL-4 on Vδ2⁻ γδT cells also occurred when IL-2 was present in the culture medium (i.e.,when the culture medium contained both IL-2 and IL-4). These dataconfirmed that the inhibitory effects of IL-4 are dominant over theactivating effects of IL-2 on cultured TCRγδ⁺ T cells, and highlightedthe importance of removing IL-4 on the second culture step.

As previously explained, although many different concentrations andcombinations of IL-2, IL-7 and IL-15 were tested in parallel, IFN-γ wasconsistently found to be a much more effective reagent for promoting theselective expansion of Vδ1⁺ T cells in culture (when used in thepresence of IL-4 and a T cell mitogen, such as anti-CD3 mAb). As it waspreviously suggested (but not formally shown), the use of IFN-γ alonewas more effective (in promoting cell expansion in culture), than thecombination of IFN-γ with either IL-2, IL-7 or IL-15, or than thecombination of IL-2 with either IL-15 or IL-7 (Table 12). These dataconfirmed that IL-15, IL-2 and IL-7 have a detrimental effect on TCRγδ⁺T cell expansion, when cells are cultured in the presence of IL-4 andIFN-γ.

As previously explained, fold expansions in large cell culture bagswere, as expected, of lower magnitude than those from 96-well plates,but still generated relevant numbers for clinical translation. Totalabsolute cell numbers obtained after large-scale cell culture inclinical grade cell bags are now detailed in Table 13.

As previously explained, the cell culture protocol obtained with thepreviously described method is appropriate for use in clinicalapplications. In fact, several materials and reagents have been approvedby at least one regulatory agency (such as the European Medicines Agencyor the Food and Drug Administration) for use in clinical applications.The full list is detailed in Table 14.

As previously described, the 2-step method of cell isolation proposed bythe described invention generates cell samples enriched in TCRγδ⁺ Tcells that are viable and can be further cultured. FIG. 17 shows in moredetail FACS-plots of TCRγδ⁺ PBL enrichment after two-step MACS-sorting.

As previously explained, a very reproducible expansion was achieved withthe culture method of the present invention, and the composition of thefinal cellular product was remarkably similar across multiple donors.

For a more complete characterization of cells obtained with thepreviously described invention, and given the novelty of the method andresulting cellular product, we performed large-spectrum phenotyping of332 different cell surface markers (FIG. 18). Vδ1⁺ T cells were comparedat the beginning (day 0) and the end (day 21) of the cell cultureprocess. We observed marked upregulation of the activation markers CD69and CD25 and HLA-DR, as well as the costimulatory receptors CD27,CD134/OX-40 and CD150/SLAM, indicators of enhanced proliferativepotential of in vitro-generated Vδ1⁺ T cells (compared to their baselineVδ1⁺ T cell counterparts). Moreover, expanded Vδ1⁺ T cells increased theexpression of NK cell-associated activating/cytotoxicity receptors,namely NKp30, NKp44, NKG2D, DNAM-1 and 2B4, all previously shown to beimportant players in tumor cell targeting. By contrast, key inhibitoryand exhaustion-associated molecules such as PD-1, CTLA-4 or CD94, wereexpressed either at very low levels or not expressed at all,demonstrating a striking “fitness” of expanded and differentiated TCRγδ⁺T cells even after 21 days of culture under stimulatory conditions.Notably, the upregulation of multiple molecules involved in celladhesion (e.g., CD56, CD96, CD172a/SIRPα, Integrin-β7 and ICAM-1) andchemokine receptors (CD183/CXCR3, CD196/CCR6, and CX3CR1) suggested highpotential to migrate and recirculate between blood and tissues. Of note,IL-18Rα and Notch1, which are known to promote type 1(interferon-γ-producing) responses, were also highly expressed byexpanded and differentiated TCRγδ⁺ T cells. Importantly, in support ofthe robustness of our method, we found strikingly similar cellphenotypes across all 4 tested donors, as illustrated by the heatmap(FIG. 18B). These data collectively characterize expanded anddifferentiated TCRγδ⁺ T cells as a highly reproducible cellular productof activated (non-exhausted) lymphocytes endowed with migratorypotential and natural cytotoxicity machinery.

As previously explained, preliminary experiments with the use ofblocking antibodies against activating receptors expressed on TCRγδ⁺ Tcells showed that anti-tumor cytotoxicity was partially reliant on NKG2Dand NKp30 receptors expressed by the expanded TCRγδ⁺ T cells. Additionalexperiments presented herein also revealed a role for the γδTCR in tumorcell recognition (FIG. 19).

As previously explained, the expression of activating NaturalCytotoxicity Receptors (NCRs; including NKp30 and NKp44), and NKG2D wasrobustly induced in long-term cultured Vδ1⁺ T cells. Here we show thatthe same effect was observed in the Vδ1⁻ Vδ2⁻ cell subset. When weapplied a gate (in FACS plot analysis) to the expanded (anddifferentiated) CD3±Vδ1⁻ Vδ2⁻ cell subset in the same cultures, weobserved that these cells expressed around the same levels of NCRs asexpressed by differentiated Vδ1⁺ cells (FIG. 20). These data furtherconfirmed that the 2-step protocol described in the present inventioncan expand and differentiate both Vδ1⁺ and Vδ1⁻ Vδ2⁻ TCRγδ⁺ T cellsubsets. In the first culture step, in the presence of a T cell mitogenand IL-4 (and in the absence of IL-15, IL-2 or IL-7), both Vδ1⁺ and Vδ1⁻Vδ2⁻ TCRγδ⁺ T cell subsets expanded in culture, but could notdifferentiate towards a cytotoxic phenotype. When the obtained cellswere subcultured in a second culture medium in the presence of a T cellmitogen and IL-2, or IL-7, or IL-15 (and in the absence of IL-4), bothcell subsets differentiated expressing high levels of activating NKreceptors that, in turn, mediated the killing of tumor cells.

As previously explained, expanded and differentiated Vδ1⁺ cells obtainedwith the method of the present invention were highly cytotoxic againstleukemia cells in vitro. Here we show in more detail that the expandedand differentiated Vδ1⁻ Vδ2⁻ cell subset is also highly cytotoxicagainst tumor targets. We sorted CD3±Vδ1⁺ cells and CD3±Vδ1⁻ Vδ2⁻ cellsfrom the same cultured cell samples by flow cytometry and co-culturedeach subset with target tumor cells, in vitro. We observed that bothsubsets could efficiently eliminate target cells. (FIG. 21).

TABLE 11 Fold NKp30+ Vδ1⁺ increase Vδ1⁺ Cond. Condition: T cells of Vδ1⁺T cells Exp. No (cultured 1 million cells/ml for 14 days in 96-wellplates) (%) T cells (%) 1 1 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-431.6 77 0.5  2* 20 ng/ml IL-2 + 1 μg/ml α-Vδ1 TCR mAb 4.7  8 6.2 3 500ng/ml IL-2 + 1 μg/ml PHA 10.5  4 13.4  4* 20 ng/ml IL-2 + 1 μg/ml α-CD3mAb 5.3  2 9.1 5 20 ng/ml IL-2 + 1 μg/ml PHA (control) 1.9  1 10.6 2 120 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 51.7 75 0.0 2 20 ng/mlIL-2 + 1 μg/ml α-Vδ1 TCR mAb + 20 ng/ml IL-4 55.9 69 0.2 3 5 ng/mlIL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 61.9 62 0.0  4* 1 μg/ml PHA + 20ng/ml IL-4 79.6 38 0.3 5 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4(previous best) 45.3 27 0.2 6 100 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/mlIL-4 55.7 22 0.4 7 300 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 57.0 211.6  8* 20 ng/ml IL-2 + 20 ng/ml IL-4 2.4  2 0.0 3 1 5 ng/ml IL-15 + 1μg/ml α-CD3 mAb + 20 ng/ml IL-4 61.4 138  0.3 2 5 ng/ml IL-2 + 1 μg/mlα-CD3 mAb + 20 ng/ml IL-4 59.4 124  1.2 3 20 ng/ml IL-2 + 1 μg/ml α-CD3mAb + 20 ng/ml IL-4 (prev. best) 45.4 105  1.0 4 5 ng/ml IL-15 + 1 μg/mlPHA + 20 ng/ml IL-4 76.2 21 1.2 5 5 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/mlIL-4 69.1 19 1.6 6 20 ng/ml IL-15 + 1 μg/ml PHA + 20 ng/ml IL-4 69.9 131.3 7 20 ng/ml IL-2 + 1 μg/ml PHA + 20 ng/ml IL-4 67.7 11 1.0 4 1 20ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 79.5 1 349   0.8 2 3ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 67.4 1 014   0.4 3 2ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 81.6 909  1.8 4 5 ng/mlIL-15 + 1 μg/ml α-CD3 mAb + 20 ng/ml IL-4 (prev. best) 69.4 624  1.9

TABLE 12 Total Fold Live Vδ1⁺ increase Cond. Condition: cells T cells ofVδ1⁺ No (cultured 1 million cells/ml for 14 days in 96-well plates) (%)(%) T cells 1 20 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 85.980.1 12 166  2 7 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 89.993.0 10 757  3 2 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100 ng/ml IL-4 87.375.2 9 394 4 0.3 ng/ml IL-15 + 2 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 100ng/ml IL-4 77.1 59.7 4 361 5 2 ng/ml IL-15 + 2 ng/ml IFN-γ + 1 μg/mlα-CD3 mAb + 100 ng/ml IL-4 90.0 67.0   811 6 0.3 ng/ml IL-15 + 1 μg/mlα-CD3 mAb + 100 ng/ml IL-4 89.7 75.5   614 7 7 ng/ml IFN-γ + 1 μg/mlα-CD3 mAb + 60 ng/ml IL-4 85.7 83.2 10 083  8 2 ng/ml IL-2 + 2 ng/mlIL-15 + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 87.5 59.4 7 208 9 2 ng/mlIL-2 + 7 ng/ml IFN-γ + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 80.4 71.6 7 15110 7 ng/ml IL-7 + 2 ng/ml IL-15 + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 91.165.6 6 193 11 2 ng/ml IL-2 + 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 88.7 70.55 192 12 1 μg/ml α-CD3 mAb + 60 ng/ml IL-4 89.0 68.3 1 890 13 0.3 ng/mlIFN-γ + 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 82.7 59.9 6 139 14 2 ng/mlIL-7 + 0.3 ng/ml IFN-γ + 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 87.6 72.9 5290 15 0.3 ng/ml IL-15 + 0.3 ng/ml IFN-γ + 2 μg/ml α-CD3 mAb + 100 ng/mlIL-4 85.9 80.1 4 840 16 0.3 ng/ml IL-15 + 2 μg/ml α-CD3 mAb + 100 ng/mlIL-4 90.0 64.2 2 943 17 2 μg/ml α-CD3 mAb + 100 ng/ml IL-4 85.8 73.0 1826

TABLE 13 Total live cells generated from 1 Buffy Coat unit: (millions ofcells) Donor: Day 0 Day 21 A 2.4  968.0 B 4.8 1 004.0 C 83.3  440.0 D5.7 1 152.0 E 9.2 1 024.0 F 25.0 1 564.0 G 4.0 1 604.0 H 2.0 1 276.0

TABLE 14 Product Manufacturing Reagent/Material Manufacturer referencequality system* For magnetic depletion of TCRα/β⁺ cells: CliniMACS ®Plus Instrument Miltenyi Biotec, GmbH 151-01 cGMP, ISO 13485 CliniMACS ®TCRα/β Kit 200-070-407 compliant CliniMACS ® Depletion Tubing Set 261-01CliniMACS ® PBS/EDTA Buffer 700-25 For magnetic enrichment of CD3⁺cells: CliniMACS ® CD3 reagent Miltenyi Biotec, GmbH 273-01 cGMP, ISO13485 CliniMACS ® Tubing Set TS 161-01 compliant For cell culture: Cellculture cassettes Saint-Gobain CC-0500 cGMP, 21 CFR Clamps Saint-Gobain1C-0022 820 compliant VueLife ® cell culture FEP bag Saint-Gobain 750-C1OpTmizer ™ T-cell expansion medium Thermo Fisher Scientific A10485-01cGMP, ISO L-Glutamine Thermo Fisher Scientific 25030-032 13485: 2003Human anti-CD3 mAb (clone OKT-3) Miltenyi Biotec, GmbH 170-076-116 orISO Recombinant Human IL-4 CellGenix GmbH 1003-050 9001: 2008Recombinant Human IL-21 CellGenix GmbH 1019-050 compliant RecombinantHuman IFN-γ R&D Systems 285-GMP Recombinant Human IL-1β CellGenix GmbH1011-050 Recombinant Human IL-15 CellGenix GmbH 1013-050 *Note: Someproducts are sold as certified medical devices for use in the EU and/orUS. All other products are sold for the manufacturing of cell-basedproducts for clinical research. They can be used in clinical trialsunder Investigational New Drug (IND) or Investigational Device Exemption(IDE) applications.

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1. A method for expanding Vδ2⁻ TCRγδ⁺ T cells in a sample comprising:(1) culturing cells in the sample in a first culture medium comprising aT cell mitogen and a growth factor having interleukin-4-like activity;in the absence of a growth factor having interleukin-15-like activity;and (2) culturing the cells obtained in step (1) in a second culturemedium comprising a T cell mitogen and a growth factor havinginterleukin-15-like activity, in the absence of a growth factor havinginterleukin-4-like activity.
 2. A method according to claim 1 whereinthe growth factor having interleukin-4-like activity is interleukin-4.3. A method according to claim 1 wherein the growth factor havinginterleukin-15-like activity is either interleukin-15, interleukin-2, orinterleukin-7.
 4. (canceled)
 5. A method according to claim 1 whereinthe first or second culture medium, or both culture media, furthercomprise a second growth factor.
 6. A method according to claim 5wherein said growth factor is interferon-γ or a mimetic or functionalequivalent thereof.
 7. A method according to claim 1 wherein the firstor second culture medium, or both culture media, further comprise asecond and a third growth factors.
 8. A method according to claim 7wherein said growth factors are interferon-γ and interleukin-21 or amimetic or functional equivalent thereof.
 9. A method according to claim1 wherein the first or second culture medium, or both culture media,further comprise a second, a third and a fourth growth factor.
 10. Amethod according to claim 9 wherein said growth factors areinterferon-γ, interleukin-21 and interleukin-1β or a mimetic orfunctional equivalent thereof.
 11. A method according to claim 1 whereinthe first or second culture medium, or both culture media, furthercomprise a costimulatory molecule of Vδ2⁻ TCRγδ⁺ T cells.
 12. A methodaccording to claim 11 wherein the costimulatory molecule is either amolecular ligand or an agonist of SLAM receptor, a molecular ligand oran agonist of CD27 receptor, or a molecular ligand or an agonist of CD7receptor.
 13. A method according to claim 1 wherein the first and secondculture media further contain serum or plasma.
 14. A method according toclaim 1 wherein prior to step (1) the cells in the sample are enrichedfor T cells; enriched for TCRγδ⁺ T cells; depleted of TCRαβ⁺ T cells;first depleted of TCRαβ⁺ T cells, and then enriched for CD3⁺ cells; ordepleted of non-TCRγδ⁺ T cells. 15-18. (canceled)
 19. A method accordingto claim 1 wherein the sample is blood or tissue or fractions thereof.20. A method according to claim 19 wherein the sample is selected fromperipheral blood, umbilical cord blood, lymphoid tissue, epithelia,thymus, bone marrow, spleen, liver, cancerous tissue, infected tissue,lymph node tissue or fractions thereof.
 21. (canceled)
 22. A methodaccording to claim 1 wherein the sample consists of low densitymononuclear cells (LDMCs) or peripheral blood mononuclear cells (PBMCs).23. A method according to claim 1 wherein in the first culture mediumthe T cell mitogen is present in an amount from about 10 to about 5000ng/ml and the growth factor having interleukin-4-like activity ispresent in an amount from about 1 to about 1000 ng/ml. 24-26. (canceled)27. A method according to claim 1 wherein in the second culture mediumthe T cell mitogen is present in an amount from about 0.1 to about 50μg/ml and the growth factor having interleukin-15-like activity ispresent in an amount from about 1 to about 1000 ng/ml. 28-30. (canceled)31. A method according to claim 1 wherein in the first or second culturemedium, or in both culture media, the growth factor havinginterferon-γ-like-activity is present in an amount from about 1 to about1000 ng/ml; and the growth factor having interleukin-21-like-activityand the growth factor having interleukin-1β-like activity are present inan amount from 1 to about 500 ng/ml. 32-34. (canceled)
 35. A methodaccording to any one of the preceding claims wherein the T cell mitogenis an antibody or a fragment thereof.
 36. A method according to claim 35wherein the antibody binds to CD3 or a fragment thereof.
 37. A methodaccording to claim 13 wherein the serum or plasma is present in anamount from about 0.5 to about 25% by volume. 38-40. (canceled)
 41. Acell preparation enriched in TCRγδ⁺ T cells prepared according to themethod of claim
 1. 42-44. (canceled)
 45. A cell preparation according toclaim 1 which comprises both Vδ1⁺ TCRγδ⁺ T cells and Vδ2⁺ TCRγδ⁺ Tcells.
 46. A cell preparation according to claim 45 which comprisesabout 55-90% Vδ1⁺ TCRγδ⁺ T cells and about 1-10% Vδ2⁺ TCRγδ⁺ T cells, ofthe total TCRγδ⁺ T cells in the preparation.
 47. (canceled)
 48. Apharmaceutical composition comprising TCRγδ⁺ T cells prepared accordingto the method of claim
 1. 49-51. (canceled)
 52. A vaccine comprisingTCRγδ⁺ T cells prepared according to the method of claim
 1. 53-58.(canceled)
 59. A method of modulating an immune response comprisingadministering an effective amount of TCRγδ⁺ T cells obtained accordingto the method claim 1 to an animal in need thereof.
 60. A method fortreating an infection comprising administering an effective amount ofTCRγδ⁺ T cells obtained according to the method of claim 1 to an animalin need thereof.
 61. A method for treating cancer comprisingadministering an effective amount of TCRγδ⁺ T cells obtained accordingto the method of claim 1 to an animal in need thereof.
 62. The methodaccording to claim 60, wherein the cancer is chronic lymphocyticleukemia, chronic myelogenous leukemia, acute myelogenous leukemia,acute lymphoblastic leukemia, and T cell and B cell leukemias, lymphomas(Hodgkin's and non-Hodgkins), lymphoproliferative disorders,plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, carcinomasof solid tissues, hypoxic tumors, squamous cell carcinomas,genitourinary cancers such as cervical and bladder cancer, hematopoieticcancers, head and neck cancers, and nervous system cancers.
 63. A methodfor vaccinating an animal comprising administering an effective amountof TCRγδ⁺ T cells obtained according to the method of claim 1 to ananimal in need thereof.